Recombinant Expression Vector Elements (REVES) for Enhancing Expression of Recombinant Proteins in Host Cells

ABSTRACT

Compositions and methods comprising recombinant expression vector elements (rEVEs) to enhance the level of expression of recombinant proteins are described. Other compositions and methods for lowering, substantially suppressing, or essentially silencing expression of a recombinant protein are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/079,748, filedMar. 28, 2008 (now U.S. Pat. No. 7,935,808), which claims the benefit ofpriority to U.S. provisional application No. 60/921,141, filed Mar. 30,2007, now expired.

BACKGROUND

A variety of systems are available that employ nucleic acid vectormolecules for the expression of recombinant proteins in a diverse andwide variety of eukaryotic or prokaryotic host cells. The decision ofwhich vector/host cell pair to use typically depends upon which systemprovides the highest yield of the desired recombinant gene product in aform best suited for its intended use. For example, if apost-translational modification, such as glycosylation, is critical torequirements of the desired recombinant protein (e.g., for antigenicity,activity, conformation, etc.), then a eukaryotic host cell system willbe desired as prokaryotic cells are characteristically devoid ofpost-translational glycosylation activity. It is also important that avector/host cell pair not only produce an expressed gene product in thedesired form, but also that molecules of the desired expressed geneproduct can be readily isolated from the cells that produce them.

Owing to the escalating costs involved in the development of recombinantproteins intended for human therapy, there remains an ongoing effort toenhance the level of expression of such recombinant proteins, especiallyin systems that employ eukaryotic host cells. Various cis- andtrans-acting regulatory elements have been characterized that havenucleotide sequences (DNA or RNA) that may improve efficiency ofexpression and/or overall yield of the desired recombinant gene product.Such regulatory elements include, but are not limited to, highlyefficient promoters, transcriptional enhancer sequences (see, e.g., thereview by Dillon and Grosveld, Trends Genet., 9: 134 (1993), locuscontrol regions (LCRs; see, e.g., Grosveld et al., Cell, 51: 975(1987)), matrix or scaffold attachment regions (MARs, SARs; see, e.g.,U.S. Pat. No. 7,129,062; Bode et al., Int. Rev. Cytol., 162A: 389-454(1995); Bode et al., Crit. Rev. Eukaryotic Gene Expression, 6: 115-138(1996)); insulator elements (see, e.g., Kellum et al., Cell, 64: 941(1991); and internal ribosome entry sites (IRES; see, e.g., review byMcBratney et al., Curr. Opin. Cell Biol., 5: 961 (1993)). Some of thenucleic acid sequences of such elements have been subsequently found topossess specific subsequences that can affect expression.

Despite advances in the understanding of various sequences and otherfactors that can affect expression of recombinant proteins in varioustypes of host cells, needs remain to improve the yields of recombinantproteins, particularly when such recombinant proteins are intended fortherapeutic or other specialized applications.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules(polynucleotide molecules) comprising nucleotide base sequences thatenhance expression of recombinant proteins. Such nucleic acid moleculesare referred to herein as recombinant expression vector elements(rEVEs). The presence of a rEVE on an expression vector enhances thelevel of expression of one or more recombinant proteins encoded by oneor more functional genes that reside on the expression vector ascompared to the level of expression in the absence of the rEVE. SuchrEVE-mediated enhancement of the level of expression of a recombinantprotein is possible whether a rEVE is located 5′ to, 3′ to, or both 5′and 3′ to (e.g., flanking) a gene encoding a recombinant protein(s) ofinterest. A rEVE present on an expression vector may enhance expressionof one or more recombinant proteins whether encoded on separatecorresponding genes or encoded on a single polycistronic gene present onthe expression vector. REVEs may be used to enhance the level ofexpression of a recombinant protein using both stable expression systemsand transient expression systems.

In one embodiment, the invention provides an isolated rEVEpolynucleotide molecule that comprises a nucleotide base sequenceselected from the group consisting of the sequence of bases of SEQ IDNO:1 (“ARM1”), the sequence of bases of SEQ ID NO:2 (“ARM2”), and anexpression enhancing portion of any of the preceding sequences, whereinwhen the rEVE polynucleotide is present on the same expression vector asa recombinant gene encoding a recombinant protein of interest, the levelof expression of the recombinant protein will be enhanced compared tothe level of expression in the absence of the rEVE.

Preferred rEVE molecules of the invention include a 2329 base pair (bp)rEVE nucleic acid molecule that has the nucleotide base sequence of SEQID NO:1 and a 2422 by rEVE nucleic acid molecule that has the nucleotidebase sequence of SEQ ID NO:2.

The 3′ terminal region of the sequence of SEQ ID NO:2 contains sequencesthat are critical for rEVE-mediated enhancement of protein expression.Such preferred 3′ terminal region sequences of SEQ ID NO:2 include thesequence of bases 462-2422 of SEQ ID NO:2 and the sequence of bases1087-2422 of SEQ ID NO:2.

An isolated rEVE nucleic acid molecule comprising one or more of thenucleotide base sequences described herein may have any of a variety offorms including, without limitation, a linear nucleic acid molecule, aplasmid, a eukaryotic viral molecule, a prokaryotic viral(bacteriophage) molecule, an artificial chromosome, and a recombinantchromosome.

In a preferred embodiment, the invention provides an expression vectorcomprising at least one rEVE described herein. Such a rEVE-containingexpression vector provides enhanced (elevated) levels of expression inan appropriate host cell of at least one recombinant protein encoded onthe expression vector compared to the level of expression in the hostcell carrying the same expression vector lacking the rEVE. Expressionvectors useful in the invention include any nucleic acid vector moleculethat can be engineered to encode and express one or more recombinantproteins in an appropriate (homologous) host cell. Such expressionvectors include, without limitation, eukaryotic plasmid vectors,eukaryotic viral vectors, prokaryotic plasmids, bacteriophage vectors,shuttle vectors (e.g., a vector that can replicate in eukaryotic andprokaryotic cells), mini-chromosomes, and various artificialchromosomes. Preferably, an expression vector is a plasmid expressionvector, more preferably, a plasmid expression vector that stablyintegrates into a eukaryotic host cell genome, and even more preferably,a plasmid expression vector that stably integrates into a host cellgenome by non-homologous recombination.

In another embodiment, the invention provides a host cell that containsan expression vector comprising a rEVE described herein and arecombinant gene that directs the expression of at least one recombinantprotein in the host cell. A host cell may be a eukaryotic or prokaryotichost cell. Preferred eukaryotic host cells for use in the inventioninclude, without limitation, mammalian host cells, plant host cells,fungal host cells, eukaryotic algal host cells, protozoan host cells,insect host cells, and fish host cells. More preferably, a host celluseful in the invention is a mammalian host cell, including, but notlimited to, a Chinese hamster ovary (CHO) cell, a COS cell, a Vero cell,an SP2/0 cell, an NS/0 myeloma cell, a human embryonic kidney (HEK 293)cell, a baby hamster kidney (BHK) cell, a HeLa cell, a human B cell, aCV-1/EBNA cell, an L cell, a 3T3 cell, a HEPG2 cell, a PerC6 cell, andan MDCK cell. Particularly preferred is a CHO cell that can be treatedwith a standard methotrexate treatment protocol to amplify the copynumber of recombinant genes on an expression vector inserted into thehost cell. Fungal cells that may serve as host cells in the inventioninclude, without limitation, Ascomycete cells, such as Aspergillus,Neurospora, and yeast cells, particularly yeast of a genus selected fromthe group consisting of Saccharomyces, Pichia, Hansenula,Schizosaccharomyces, Kluyveromyces, Yarrowia, and Candida. Preferredyeast species that may serve as host cells for expression of recombinantproteins according to the invention include, but are not limited to,Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis,Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.Prokaryotic host cells that may be used for expressing recombinantproteins according to the invention include, without limitation,Escherichia coli, serovars of Salmonella enterica, Shigella species,Wollinella succinogenes, Proteus vulgaris, Proteus mirabilis,Edwardsiella tarda, Citrobacter freundii, Pasteurella species,Haemophilus species, Pseudomonas species, Bacillus species,Staphyloccocus species, and Streptococcus species. Other cells that maybe used as host cells for expression of recombinant proteins accordingto the invention include protozoan cells, such as the trypanosomatidhost Leishmania tarentolae, and cells of the nematode Caenorhaditiselegans.

REVE polynucleotides as described herein, vectors comprising one or morerEVEs described herein, and host cells comprising such vectorscomprising one or more rEVEs as described herein may be used in avariety methods related to expression of recombinant proteins ofinterest.

In one embodiment, the invention provides a method of enhancingexpression of a recombinant protein of interest in a host cellcomprising the step of inserting into a host cell a recombinantexpression vector that comprises a rEVE described herein and arecombinant gene that encodes and directs the synthesis of therecombinant protein of interest in the host cell and culturing the hostcell under conditions promoting expression of the recombinant protein.

In another embodiment, the invention provides a method of producingmethotrexate-resistant host cells that stably (as opposed totransiently) express a recombinant protein at an elevated level in theabsence of methotrexate (“MTX”), i.e., in the absence of the selectivepressure for elevated expression of the recombinant protein provided bythe presence of methotrexate. Such a method comprises the step ofinserting into host cells an expression vector that comprises arecombinant gene encoding a recombinant protein of interest, a rEVEdescribed herein, and a gene encoding a dihydrofolate reductase(“DHFR”); growing the host cells in the presence of methotrexate toselect for a methotrexate-resistant host cell that expresses therecombinant protein of interest; and isolating a methotrexate-resistanthost cell, wherein the methotrexate resistant host cell expresses therecombinant protein of interest at a level that is higher than that of amethotrexate-sensitive host cell, and wherein the methotrexate-resistanthost cell stably expresses an elevated level of the recombinant proteinof interest when grown in the presence or in the absence ofmethotrexate. In a particularly preferred embodiment, the rEVE used inthis method comprises the sequence of SEQ ID NO:2 or an expressionenhancing portion thereof that, when present on the same expressionvector as a gene encoding a recombinant protein of interest, enhancesthe level of expression of the recombinant protein in a host cell.

In another embodiment, the invention provides a method for producing apopulation of host cells with enhanced or improved adaptation to thepresence of methotrexate in a DHFR-methotrexate procedure for amplifyingrecombinant protein expression comprising the step of inserting intohost cells an expression vector comprising a gene encoding a recombinantprotein of interest, a rEVE described herein, and a DHFR gene, wherein apopulation of the host cells containing the expression vector adaptsbetter, i.e., has a higher survivability and/or higher growth rate, inthe presence of methotrexate compared to a population of host cellscarrying the expression vector lacking the rEVE sequence.

In yet another embodiment, the invention provides a method of enhancingthe amplification (elevation) of expression of a recombinant protein inhost cells obtained using a DHFR-methotrexate procedure comprising thestep of inserting into host cells an expression vector comprising arecombinant gene encoding a recombinant protein of interest, a rEVEdescribed herein, and a DHFR gene; growing the host cells in thepresence of methotrexate to select for a methotrexate-resistant hostcell that expresses the recombinant protein of interest; and isolatingan methotrexate-resistant host cell, wherein the isolatedmethotrexate-resistant host cell expresses the recombinant protein ofinterest in the presence of methotrexate at a level that is higher thanthat of a methotrexate-resistant host cell containing the sameexpression vector lacking a rEVE.

Methotrexate may be conveniently employed in methods described herein ina range of 20 nM to 500 nM, although lower and higher concentrations,such as 5 nM to 10 μM, may also be successfully employed in such methodsto select for amplified expression of recombinant proteins of interest.

In still another embodiment, the invention provides a method oflowering, substantially suppressing, or essentially silencing expressionof a recombinant protein from an expression vector. Such methods employexpression vectors that comprise one or more fragments of a rEVE thatprovides lower levels of expression of a particular recombinant geneproduct than provided using a rEVE comprising the sequence of SEQ IDNO:1 or SEQ ID NO:2. Such fragments have a particular truncated variantof SEQ ID NO:2, including a nucleotide base sequence of bases 1-1086 ofSEQ ID NO:2 or of bases 1-461 of SEQ ID NO:2. A nucleic acid moleculehaving the truncated ARM2 sequence consisting of bases 1-461 of SEQ IDNO:2 is particularly useful in suppressing or substantially silencingexpression of a recombinant protein from a vector molecule in a hostcell. These findings also indicate that the sequence of nucleotide bases462-2422 of SEQ ID NO:2 and the sequence of nucleotide bases 1087-2422of SEQ ID NO:2 are most preferred for maximal rEVE-mediated enhancementof expression of recombinant proteins.

A recombinant protein whose expression may be enhanced by one or morerEVE molecules described herein may be any protein (including peptides,polypeptides, and oligomeric proteins) for which a functional gene(s)can be engineered into a nucleic acid vector molecule for expression inan appropriate host cell. Such proteins include, without limitation,soluble proteins, membrane proteins, structural proteins (i.e., proteinsthat provide structure or support to cells, tissues, or organs),ribosomal proteins, enzymes, zymogens, antibody molecules, cell surfacereceptor proteins, transcription regulatory proteins, translationregulatory proteins, chromatin proteins (e.g., histones), hormones, cellcycle regulatory proteins, G proteins, neuroactive peptides,immunoregulatory proteins (e.g., interleukins, cytokines), bloodcomponent proteins, ion gate proteins, heat shock proteins,dihydrofolate reductase, an antibiotic resistance protein, functionalfragments thereof, epitope-containing fragments thereof, andcombinations thereof.

Nucleic acid molecules containing a sequence of a rEVE described hereinor portion thereof may also be used in as nucleic acid probes foridentifying the presence of rEVE sequences in other nucleic acidmolecules by nucleic acid hybridization or as a source of primers foruse in various polymerase chain reaction (PCR) procedures, e.g., as maybe employed for manipulating, identifying, producing, or amplifying rEVEsequences described herein.

REVE sequences, such as those for ARM1 (SEQ ID NO:1) and for ARM2 (SEQID NO:2), may comprise one or more matrix attachment region (MAR)sequences. MAR sequences may occur in clusters within a rEVE sequence,including in clusters at the 5′ and/or 3′ terminal regions of a rEVEsequence. REVE polynucleotides as described herein are a useful sourceof MAR sequences. Accordingly, the invention provides compositions andmethods that are useful for increasing MAR sequences in a nucleic acidmolecule comprising inserting into the nucleic acid molecule a rEVEdescribed herein or a portion of a rEVE described herein containing oneor more MAR sequences.

Nucleotide base sequences described herein also serve to provide thecomplementary sequences thereof. DNA molecules and nucleotide basesequences described herein also provide the corresponding RNA moleculesand base sequences, wherein thymine (T) is replaced by uracil (U), andnucleic acid sequences complementary thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of plasmid expression vector pA205 usedto express the immunoglobulin heavy and kappa light chains that form anactive human anti-TNF-α IgG1 molecule (“adalimumab”) in stabletransfectants of Chinese hamster ovary (CHO) cells. Abbreviations:“ENHANCER” refers to the intermediate early gene enhancer ofcytomegalovirus (CMV); “ADENO PROMOTER” refers to the major latepromoter of adenovirus; “ADALIMUMAB HEAVY CHAIN” refers to the codingregion for IgG1 heavy chain of adalimumab; “SV40 Poly A” refers to thesimian virus 40 polyadenylation site; “GASTRIN TERMINATOR” refers to thetranscription termination signal of the human gastrin gene; “SV40PROMOTER” is the simian virus 40 promoter; “DHFR” refers to mousedihydrofolate reductase gene; “TK Poly A” refers to the polyadenylationsite of the herpes simplex virus thymidine kinase gene; “ADALIMUMABLIGHT CHAIN” refers to the coding region for kappa light chain ofadalimumab; “ORI”refers to the plasmid Co1E1 prokaryotic origin ofreplication that functions in Escherichia coli; “P(BLA)” refers to theprokaryotic promoter of the β-lactamase gene (ampicillin resistancegene) and small arrow indicates direction of transcription; “APr”(“ampicllin resistance”) refers to the coding region for β-lactamase.Arrows indicate direction of transcription (5′ to 3′).

FIG. 2 is a schematic diagram of plasmid expression vector pA205Genomicin which ARM1 (“A1”) nucleic acid that has the nucleotide base sequenceof SEQ ID NO:1 is inserted into expression vector pA205 (FIG. 1)upstream of the adenovirus major late promoter and adalimumab IgG1 heavychain coding region, and ARM2 (“A2”) nucleic acid that has thenucleotide base sequence of SEQ ID NO:2 is inserted downstream of theadalimumab kappa light chain coding region. For other abbreviations, seethe description of FIG. 1, above.

FIG. 3 is a schematic diagram of plasmid expression vector pA205A1 inwhich ARM1 (“A1”) nucleic acid having the sequence of SEQ ID NO:1 isinserted into expression vector pA205 (FIG. 1) upstream of the adenopromoter and the adalimumab IgG1 heavy chain coding region. For otherabbreviations, see the description of FIG. 1, above.

FIG. 4 is a schematic diagram of plasmid expression vector pA205A2 inwhich ARM2 (“A2”) nucleic acid having the nucleotide base sequence ofSEQ ID NO:2 is inserted downstream of the adalimumab kappa light chaincoding region. For other abbreviations, see the description of FIG. 1,above.

FIG. 5 is a schematic diagram of plasmid expression vectorpCHOEL246GGhum used to express the immunoglobulin heavy and kappa lightchains that form an active human anti-EL-selectin IgG1 molecule instable transfectants of Chinese hamster ovary (CHO) cells.“ANTI-EL-SELECTIN HEAVY CHAIN” refers to the coding region for the heavychain of a human anti-EL-selectin IgG1 antibody; “ANTI-EL-SELECTIN LIGHTCHAIN” refers to the coding region for the kappa light chain of a humananti-EL-selectin IgG1 antibody. For other abbreviations, see thedescription of FIG. 1, above.

FIG. 6 is a schematic diagram of plasmid expression vectorpA-Gen-EL-Selectin in which ARM1 (“A1”) nucleic acid having thenucleotide base sequence of SEQ ID NO:1 is inserted into expressionvector pCHOEL246GGhum (FIG. 5) upstream of the anti-EL-selectin IgG1heavy chain coding region, and ARM2 (“A2”) nucleic acid having thenucleotide base sequence of SEQ ID NO:2 is inserted downstream of theanti-EL-selectin kappa light chain coding region. For otherabbreviations, see the description of FIG. 1, above.

FIG. 7 is a schematic diagram of plasmid expression vector pA205-eGFPused to express enhanced green fluorescent protein (eGFP) in stabletransfectants of CHO cells. The eGFP coding region (“EGFP”) is inserteddownstream from the adenovirus major late promoter (“ADENO PROMOTER”)into pA205 (FIG. 1) from which the light chain coding region, proximalpromoter, and proximal enhancer and the heavy chain coding region havebeen deleted. For other abbreviations, see the description of FIG. 1,above.

FIG. 8 is a schematic diagram of plasmid expression vector pA205G-eGFPin which ARM1 (“A1”) nucleic acid having the nucleotide base sequence ofSEQ ID NO:1 is inserted into expression vector pA205-eGFP (FIG. 7)upstream of the eGFP coding region (“EGFP”), and ARM2 (“A2”) nucleicacid having the nucleotide base sequence of SEQ ID NO:2 is inserteddownstream of the eGFP coding sequence. For other abbreviations, see thedescription of FIG. 1, above.

FIG. 9 is a schematic diagram of plasmid expression vectorpA205A2-Spe-trunc, which is essentially identical to expression vectorpA205A2 shown in FIG. 4, except that the ARM2 (SEQ ID NO:2) has beenreplaced with a truncated ARM2 variant, “A2 (bp 1-1086)”, which has thenucleotide base sequence of bases 1-1086 of SEQ ID NO:2 as the result ofdigestion of ARM2 nucleic acid with restriction endonuclease SpeI. Forother abbreviations, see the description of FIG. 1, above.

FIG. 10 is a schematic diagram of plasmid expression vectorpA205A2-Swa-trunc, which is essentially identical to expression vectorpA205A2 shown in FIG. 4, except that ARM2 (SEQ ID NO:2) has beenreplaced with a truncated ARM2 variant, “A2 (bp 1-461)”, which has thenucleotide base sequence of bases 1-461 of SEQ ID NO:2 as the result ofdigestion of ARM2 nucleic acid with restriction endonuclease Swa1. Forother abbreviations, see the description of FIG. 1, above.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the discovery that one or more isolatednucleic acid molecules (polynucleotide molecules) comprising anucleotide sequence of SEQ ID NO:1:

aattgaattcgttccctttagtgagggttaattccgcggccgcgtcgacagctctagagggagtgccaggataggctccaaagctacacagagaatccctgtttcaaaaaaccaaaaaaaaaaaaataaaaaataaaaaataaaaagtagggtacagatctaaatagacaattctcaatagaggaatctaaaatgcctgaaagacaaataagaaagtgttcaacatccttagccatcagggaaatgcaaatcaaaacaactctgagatactatcttactcctgtcataatggccaaattcaaaaacactaatgacagttcatgttggagagaatgtggagaaagaggagcacttctccactgctggtgggagtgccaacttggacagccactttggaaatcagtatggctactcctcaagaaaatggaaatcagtttaccacaagatccagcaattccactcaggcatatacccaaaagaaccgcattcatacaagcaatatctgttcaacgatgttcatagcagctctatttgtaacagccagaaactggaagcagcctagttgcacctcaaccaaagaaatggatagagaaaatatggtacatttatgcaatggagtactactcagcggaaaagtacaatggaatcttgaaatttgcaagaaaatggatggaactagaagaaacctttctgagcaaggtaactcaatcacaaaaagacaaacatgatatgtaatcactcatatgtggattttagacacagtgtaaaggattaccagcctacaatccacactgccaaagaacctaataaacaaggaggaccctaagggagacatacatggtcccctggagatggggaatgggtcaagatatgctgagcaaagtgggaacatgggaagaggggggaaggagctaggaaattgagaaagggagaaaaggagggatgcagaggacataagggagcagaaacattgactcagggaatgaatcgaagataacaagccatggagatatcataatagagggagacattttgggtatacagagaaatcaggcacttgggaaatgtctggaaatctacaaagtataacaccaggtaacaatctaagcaacagaggagaggctaccttaaatgtcctaccctgatagtgagattgatgactaacttatatgccatgttatagccttcatccagcagctggtggaagtagaagcagacacccataactaatcacggaactgaactggaacccagattcagagaaggatgagtgaagggcacagaggtccagaccaggctggtgaaacccacaaaaacagttgaactgaatatcggtgaactcttgctccccagactgatagctggaataccagcatgggactgatccagactccaggaacatgagttcctgtgaggaaacctcggaaatctaagggacctcctgtagaagttcagtacttatccctagcataggtgtggactgagggagcccattccatatagaggaatactctctggagccaacacacatgggggtgggcataggccctttcccaaagcatacaatagactcggatgacaccctatggaaggcctcatcatccagggggagcagaaaggatatgtgatagacagggtttcagttgggagccgggtagtgggaggggagaattggtggaagaaggaaaccgggattgtcatgtaaatcaatgctgtttctaattcaaataagaaagttgaaaaaaaagaaaactgatacttattgcaccatgtaatgttatgaaatggcatttgctgttaagatgagcagtctatctgctaatctccctagctggcttgtgaacttgttatatggacaaagctggtctcaaattcaaagatatttgcgtatgtctgtctcctgagtgttgagagtacaagtatgtaccaccaatccctttgattatacaattacatttgaaaacagtttgagatttaattataactatgcaatcaattcaaaataataaatttaaatctcatatttgtctttaggtggaaatctgttaatatacatcatgattatatattttaatttattatatgttttctaggacaaaatatactaaaatgaaatctaaggctctaaacatacaaaactgtatgcatagatacatcacgatcatataatttccatgacatgctattcgggaatataatgatctacctgcagtaatgattaatttggaaatgctgaatacaactgcttctcttttgaaaatacaaattccttacatttgtaatctatttaattttaaaggttgtaccccagaaagtagtgaattcttaa,or a nucleotide sequence of SEQ ID NO:2:

aagaatatgctcaatgtaatacccatggcaggcattcaatgtttgtctgtcttcatattgaagataaacagatgtatatcatatacaaaaatatttaatgtgaagttgtccatgtgttcaggatctatatactttcaaaaatctttttccatattcttttcttaatcctcctgaagtgtagaccattatactggaaaaccgtcactattgtacaggataggagcctttgactctgagaggatcccatacattgattgtattttcaaatatattttggctgcttttctccatgtgatatttggcaatctggagaggcatttgctcctggaaatttatcaatgttgacaatgttgtttacatgttttaagtaactattttgctaccaaggaaactgcttcactccctttcacatataaaactcataaaatattgaaaggctccaataagtttaaatcattctgtattgctcatggagatttaaatttcagtgctaattttttattagcactttaatttagaaggcaccaggtttctacaagatttaaaattattggagcatttcaaaattttataagctttccagtaaggttgtggctatgattctttgcttgtaaagtaaagtgcaatttaaagttaatttaaataatttaactgctgcagacattttaggagaattgtttgtatttcaaactgaaattcagggtagacaattagaataattttacaaagaggaaatatttttctaataataaattagtaactctaacttatattaaaatttaagtcctcattgctttcaatattttaacaaccctattgtattatttttcttataaatatttgaatttataatgatcaaagaatttctttgatacaagtgtctaaatgattaccatcaaactgttggtaggagcttgttatatatgtgttttaccttatgttttttgatacttcatttgttactgtactgtgatcgagttaattccctactgaaactaaaaatgctatcacatagttttagcatcatctgttggggaaatggctattttaactactctgagatgagaaattcaacaccattcactaacaatatagggaaactagtgttggtagattgttgagtgcttatacatatatcttgtcccatggttaactataagttggtgtctgttgctgccacccagtatggaaacacattatgttttttctttttttttttttatagccatgagaaagaccaaaattctatacttgaaaaaccgtttatattgaatgtgtattcctttcacgtccaccttagattcaactcctaagtcaatttatggtaaagcatagatcatctgcttgacaacagtttggatgatgatcttggaaaaaatgccttattatatgatacaatggaattaatgatatgagctgaataaatatatcaatattcaaatgacatactaatatttatgtctaagagaatgtgttcaaagtagatgaaagtgccttcacttgaaaattcatctgagttaaaacagatagttgcttcggttttagttatttcagaggtattcaagttgacaactaagaatagccgtcacagatacatatcaattatggacccaaattctattgaatgtcagctacatattcttatagaaaataggaacctagatgaggccgtgttcttggaatgaattttcaacacattgtatgagggttttattgtggttttggttgttgttttactttcctttttttccatagacaaatttgtcccatgtacccacaaggtgaccagtggtgacaagcctactccaggagtcctggtgaataaagattatacaagatagtagagactcatcaaaacaataagaaaaagagaatacatagggcagaaatttctcattttctcagctatggtatcctatttcactcttgtactattctactcactagaagtcagtgactaccataactcagtggctgtgccctagatcaaaggaaacattatttcaaggcatgaatgtcagccacaccttcatagtgggttacttttaatttgtttagtaagaatagacaccctactttggttaggaaacataaacttacaagacattcattggtttttctttactaaattaaatcattaagaaaacgtaattatcagagtttaaatggcatgaaacatagaaatactcatttgctgccctgatttattttcccaagaatattttcaatgtcttctttggaagctccttggtaaatgcactttctttcactcatttatgaggtctgtgcacatcacagtcaataaaggcctgcagtattgaatcagccatacagacataattcataacatttttctatttctcatgaatcaaatattgttattgctgtacataaaataatgaatcaaagtataggtctaga,can be engineered into expression vector molecules to enhance expressionof one or more recombinant proteins from one or more genes present onthe expression vector molecules.

An isolated polynucleotide molecule that comprises a nucleotide sequenceof SEQ ID NO:1 or SEQ ID NO:2 is referred to herein as a recombinantexpression vector element (rEVE). REVEs of the invention also include,but are not limited to, a polynucleotide molecule that comprises anexpression-enhancing portion of the sequence shown in SEQ ID NO:1(“ARM1”) or SEQ ID NO:2 (“ARM2”). Sequences from the 3′ terminal regionof the ARM2 SEQ ID NO:2, such as those having the sequence of bases462-2422 of SEQ ID NO:2 and of bases 1087-2422 of SEQ ID NO:2, areparticularly useful in providing enhanced expression of a recombinantprotein of interest. A rEVE described herein may be used in combinationwith other control sequences, regulators, and procedures currentlyavailable to increase production of recombinant proteins in host cells.

In order to more clearly describe the invention the following terms aredefined:

The term “antibody” or “antibody molecule”, as used and understoodherein, broadly refer to any immunoglobulin (Ig) molecule comprised offour polypeptide chains, two heavy (H) chains and two light (L) chains,or any functional fragment, mutant, variant, or derivation thereof,which retains the essential epitope binding features of an Ig molecule.Such mutant, variant, or derivative antibodies are known in the art andinclude the non-limiting embodiments discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (VH) and a heavy chain constant region. The heavychain constant region is comprised of three domains, CH1, CH2 and CH3.Each light chain is comprised of a light chain variable region (VL) anda light chain constant region. The light chain constant region iscomprised of one domain (CL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, referred to as framework regions (FR). Each VH and VL iscomposed of three CDRs and four FRs, arranged from amino terminus tocarboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG 3, IgG4, IgA1 andIgA2), or subclass.

The terms “antibody” and “antibody molecule” also encompass one or morefragments of an antibody that retains the ability to specifically bindto an antigen. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormulti-specific formats; specifically binding to two or more differentantigens. Examples of binding fragments encompassed within the term“antibody” include a Fab fragment, i.e., a monovalent (one binding site)fragment consisting of the VL, VH, CL, and CH1 domains; a F(ab′)₂fragment, i.e., a bivalent (two binding sites) fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; an Fdfragment consisting of the VH and CH1 domains; an Fv fragment consistingof the VL and VH domains of a single arm of an antibody; a single domainantibody (dAb) (Ward et al., Nature, 341: 544-546 (1989); Winter et al.,PCT publication WO 90/05144 A1, incorporated herein by reference), whichcomprises a single variable domain; dual variable domain (DVD)antibodies (see, e.g., PCT Publication No. WO 2007/024715); and isolatedcomplementarity determining regions (CDRs). Furthermore, although thetwo domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv), see e.g., Bird et al. Science, 242: 423-426 (1988);Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Suchsingle chain antibodies are encompassed by the terms “antibody” and“antibody molecule”. Diabodies are also encompassed by the terms“antibody” and “antibody molecule”. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger et al., Proc. Natl. Acad.Sci. USA, 90: 6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123(1994)). The terms “antibody” and “antibody molecule” also encompassdual variable domain immunoglobulin molecules, such as DVD-IG™ (AbbottLaboratories) dual variable domain immunoglobulin molecules (see, e.g.,PCT publication No. WO 2007/024715).

As used herein, “vector” refers to any genetic element capable ofserving as a vehicle of genetic transfer, expression, or replication fora foreign polynucleotide in a host cell. For example, a vector may be anartificial chromosome or a plasmid, and may be capable of stableintegration into a host cell genome, or it may exist as an independentgenetic element (e.g., episome, plasmid). A vector may exist as a singlepolynucleotide or as two or more separate polynucleotides. Vectors maybe single copy vectors or multicopy vectors when present in a host cell.Preferred vectors for use in the present invention are expression vectormolecules in which one or more functional genes can be inserted into thevector molecule, in proper orientation and proximity to expressioncontrol elements resident in the expression vector molecule so as todirect expression of one or more proteins when the vector moleculeresides in an appropriate (homologous) host cell.

Expression vectors may include, without limitation, eukaryotic plasmidvectors, eukaryotic viral vectors, prokaryotic plasmids, bacteriophagevectors, shuttle vectors (e.g., a vector that can replicate ineukaryotic and prokaryotic cells), mini-chromosomes, and variousartificial chromosomes (e.g., bacterial artificial chromosomes (BACs),yeast artificial chromosomes (YACs)). Preferably, an expression vectorused in the invention is a plasmid, more preferably, a plasmidexpression vector that stably integrates into a host cell genome, and,even more preferably, a plasmid expression vector that stably integratesinto a host cell genome by non-homologous recombination. A “shuttlevector” (or bi-functional vector) refers to any vector that canreplicate in more than one species of organism. For example, a shuttlevector that can replicate in both Escherichia coli (E. coli) andSaccharomyces cerevisiae (S. cerevisiae) can be constructed by linkingsequences from an E. coli plasmid with sequences from the yeast 2μplasmid.

Expression systems comprise an expression vector and appropriate(homologous) host cell that will express the recombinant protein(s)encoded on the expression vector. An expression system may be a stableexpression system or a transient expression system. In a stableexpression system, an expression vector stably integrates into the hostcell genome or is continuously replicated and faithfully passed on toboth daughter cells so that host cells are able to continue to expressthe recombinant protein(s) when cultured under the appropriateconditions. In a transient expression system, expression vectormolecules are not retained in both daughter cells and eventually arelost or so diminished in a growing cell culture that expression ofrecombinant protein(s) from the culture will eventually cease or be solow as to not be useful for most production purposes. Expression vectorsused in the Examples, below, are types of shuttle vectors that canreplicate to relatively high copy numbers when inserted (e.g., bytransformation) into E. coli cells and that can also be inserted (e.g.,by transfection) into and stably maintained in Chinese hamster ovary(CHO) cells to obtain stable expression of their encoded gene product(s)of interest (Kaufman et al., Molec. Cell. Biol., 5: 1750-1759 (1980)) orthat can be transiently maintained in HEK 293 cells (Durocher et al.,Nucleic Acids Res., 30: E9). Thus, a rEVE polynucleotide moleculedescribed herein may be used to enhance expression of a recombinantprotein(s) of interest in both stable and transient expression systems.

Exemplary eukaryotic vectors that may be used in the invention include,but are not limited, to viral and non-viral vectors. Viral vectorsinclude, without limitation, retroviral vectors (including lentiviralvectors); adenoviral vectors including replication competent,replication deficient, and gutless forms thereof; adeno-associated virus(AAV) vectors; simian virus 40 (SV-40) vectors; bovine papilloma virusvectors; Epstein-Barr virus vectors; herpes virus vectors, vacciniavirus vectors; Moloney murine leukemia virus vectors; Harvey murinesarcoma virus vectors, murine mammary tumor virus vectors, and Roussarcoma virus vectors. Baculovirus vectors are well known and aresuitable for expression in insect cells.

A variety of vectors suitable for expression in eukaryotic orprokaryotic cells are well known in the art, and many are commerciallyavailable. Commercial sources include, without limitation, Stratagene(La Jolla, Calif.), Invitrogen (Carlsbad, Calif.), Promega (Madison,Wis.), and Sigma-Aldrich (St. Louis, Mo.). Many vector sequences areavailable through GenBank, and additional information concerning vectorsis available on the internet via the Riken BioSource Center.

A vector molecule typically comprises at least one origin of replicationand may also comprise a gene for a “marker” or “selectable marker” bywhich the vector can be identified or selected when inserted into a hostcell. Such useful markers may, without limitation, confer resistance toantibiotics, provide functions that give a selective growth advantageover cells that lack such functions, or provide a means to easilyidentify cells that possess the vector (e.g., colorigenic system). Suchmarkers are well known in the art, and the choice of the properselectable marker(s) to use in a vector molecule will depend on whathost cell will be used and what properties are desired of the host cellcontaining the vector.

The terms “functional gene construct”, “functional gene”, and “gene”refer to a polynucleotide that contains a coding sequence for one ormore proteins that is operably linked to a promoter sequence andpossibly other transcriptional regulatory sequences to direct propertranscription of the coding sequence into messenger RNA (mRNA) and thatalso comprises any of a variety of translation regulatory sequences thatmay be necessary or desired to direct proper translation of the mRNAinto the desired protein in the intended host cell. A translationalstart codon (e.g., ATG) and a ribosome binding site are typicallyrequired in the mRNA for translation to occur in prokaryotic andeukaryotic cells. Other translation regulatory sequences that may alsobe employed, depending on the host cell, include, but are not limitedto, an RNA splice site and a polyadenylation site.

The term “recombinant” is used herein to describe altered or manipulatednucleic acids, nucleic acids isolated from the environment in which theynaturally occur, host cells transfected with or otherwise manipulated tocontain exogenous nucleic acids, or proteins expressed non-naturallythrough manipulation of isolated DNA and transformation of host cells.“Recombinant” is a term that specifically encompasses DNA moleculeswhich have been constructed in vitro using genetic engineeringtechniques, and use of the term “recombinant” as an adjective todescribe a molecule, construct, vector, cell, protein, polypeptide, orpolynucleotide specifically excludes naturally occurring such molecules,constructs, vectors, cells, proteins, polypeptides, or polynucleotides.In particular, a “recombinant protein” for the purposes of the presentinvention is a protein that is expressed by a host cell which has beenmanipulated by incorporating at least one genetic element that was notnaturally occurring in the host cell prior to expression of the protein.A protein, the coding sequence for which has been artificiallyincorporated into a host cell made capable of expressing the protein,for example by being transfected with an expression vector including thecoding sequence of the protein, is a “recombinant protein” onceexpressed by the host cell.

A “host cell” refers to any cell, i.e., any eukaryotic or prokaryoticcell, into which a vector molecule can be inserted. According to thepresent invention, preferred host cells are eukaryotic or prokaryoticcells, including, but not limited to, animal cells (e.g., mammalian,bird, and fish host cells), plant cells (including eukaryotic algalcells), fungal cells, bacterial cells, and protozoan cells. Host cellsuseful in the invention may be of any genetic construct, but arepreferably haploid or diploid cells. Preferred mammalian host cellsuseful in the invention include, without limitation, a Chinese hamsterovary (CHO) cell, a COS cell, a Vero cell, an SP2/0 cell, an NS/0myeloma cell, a human embryonic kidney (HEK 293) cell, a baby hamsterkidney (BHK) cell, a HeLa cell, a human B cell, a CV-1/EBNA cell, an Lcell, a 3T3 cell, an HEPG2 cell, a PerC6 cell, and an MDCK cell. Apreferred insect cell is Sf9. Fungal cells that may serve as host cellsin the invention include, without limitation, Ascomycete cells, such asAspergillus, Neurospora, and yeast cells, particularly yeast of thegenera Saccharomyces, Pichia, Hansenula, Schizosaccharomyces,Kluyveromyces, Yarrowia, and Candida. Particularly preferred yeastfungal species that may serve as host cells for expression ofrecombinant proteins are Saccharomyces cerevisiae, Hansenula polymorpha,Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. Preferred prokaryotic cells that may serve as hostcells in the invention include, without limitation, Escherichia coli,serovars of Salmonella enterica, Shigella species, Wollinellasuccinogenes, Proteus vulgaris, Proteus mirabilis, Edwardsiella tarda,Citrobacter freundii, Pasteurella species, Haemophilus species,Pseudomonas species, Bacillus species, Staphyloccocus species, andStreptococcus species. Other cells that may be useful host cells for theexpression of recombinant proteins according to the invention includeprotozoans, such as the trypanosomatid host Leishmania tarentolae, andcells of the nematode Caenorhaditis elegans. Various expression vectorsare available for use in the aforementioned cells.

There are a variety of means and protocols for inserting vectormolecules into cells including, but not limited to, transformation,transfection, cell or protoplast fusion, use of a chemical treatment(e.g., polyethylene glycol treatment of protoplasts, calcium treatment,transfecting agents such as LIPOFECTIN® and LIPOFECTAMINE® transfectionreagents available from Invitrogen (Carlsbad, Calif.), use of varioustypes of liposomes, use of a mechanical device (e.g., nucleic acidcoated microbeads), use of electrical charge (e.g., electroporation),and combinations thereof. It is within the skill of a practitioner inthe art to determine the particular protocol and/or means to use toinsert a particular vector molecule described herein into a desired hostcell.

Methods for “transferring nucleic acid sequence information” from onevector or other nucleic acid molecule to another are not limiting in thepresent invention and include any of a variety of genetic engineering orrecombinant nucleic acid techniques known in the art. Particularlypreferred transfer techniques include, but are not limited to,restriction digestion and ligation techniques, polymerase chain reaction(PCR) protocols (utilizing specific or random sequence primers),homologous recombination techniques (utilizing polynucleotide regions ofhomology), and non-homologous recombination (e.g., random insertion)techniques. Nucleic acid molecules containing a specific sequence mayalso be synthesized, e.g., using an automated nucleic acid synthesizer,and the resulting nucleic acid product then incorporated into anothernucleic acid molecule by any of the aforementioned methodologies.

Employing genetic engineering technology necessarily requires growingrecombinant host cells (e.g., transfectants, transformants) under avariety of specified conditions as determined by the requirements of thecells and the particular cellular state desired by the practitioner. Forexample, a host cell may possess (as determined by its geneticdisposition) certain nutritional requirements, or a particularresistance or sensitivity to physical (e.g., temperature) and/orchemical (e.g., antibiotic) conditions. In addition, specific cultureconditions may be necessary to regulate the expression of a desired gene(e.g., the use of inducible promoters), or to initiate a particular cellstate (e.g., yeast cell mating or sporulation). These varied conditionsand the requirements to satisfy such conditions are understood andappreciated by practitioners in the art.

In the context of amplifying (elevating) recombinant protein expressionin a host cell using a standard dihydrofolate reductase(DHFR)-methotrexate (MTX) amplification procedure, the terms “stability”and “stable expression” refer to the ability of a culture of cells tocontinue to express the recombinant protein at an amplified (elevated)level when grown in the absence of methotrexate, i.e., in the absence ofthe selective pressure for elevated expression provided by the presenceof methotrexate used in the amplification procedure. Thus, onceisolated, a stably transfectant host cell can express a recombinantprotein of interest in the presence or absence of methotrexate.

In the context of amplifying recombinant protein expression in a hostcell using a standard dihydrofolate reductase (DHFR)-methotrexate (MTX)amplification procedure, “enhanced adaptation” or “improved adaptation”to the presence of methotrexate refers to the higher survivabilityand/or higher growth rate in the presence of methotrexate of a cultureof host cells carrying expression vectors comprising a rEVE describedherein compared to the survivability and/or growth rate in the presenceof methotrexate of a culture of host cells carrying expression vectorsthat lacks the rEVE. “Survivability” of a population of host cellsrefers to the ability of a population of host cells to grow and toreproduce in the presence of a selective pressure (e.g., methotrexate).

“Sequence homology” is a familiar concept to practitioners in thisfield. To determine the percent homology of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a secondcomparison amino acid or nucleic acid sequence). The amino acid residuesor nucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules arehomologous at that position (i.e., as used herein amino acid or nucleicacid “homology” is equivalent to amino acid or nucleic acid “identity”).The nucleic acid sequence homology may be determined as the degree ofidentity between two sequences. The homology may be determined usingcomputer programs known in the art, such as GAP software provided in theGCG program package. See, Needleman and Wunsch, J. Mol. Biol., 48:443-453 (1970).

A composition or method described herein as “comprising” one or morenamed elements or steps is open-ended meaning that the named elements orsteps are essential, but other elements or steps may be added within thescope of the composition or method. To avoid prolixity, it is alsounderstood that any composition or method described as “comprising” (or“comprises”) one or more named elements or steps also describes thecorresponding, more limited, composition or method “consistingessentially of” (or “consists essentially of”) the same named elementsor steps, meaning that the composition or method includes the namedessential elements or steps and may also include additional elements orsteps that do not materially affect the basic and novelcharacteristic(s) of the composition or method. It is also understoodthat any composition or method described herein as “comprising” or“consisting essentially of” one or more named elements or steps alsodescribes the corresponding, more limited, and close-ended compositionor method “consisting of” (or “consists of”) the named elements or stepsto the exclusion of any other unnamed element or step. In anycomposition or method disclosed herein, known or disclosed equivalentsof any named essential element or step may be substituted for thatelement or step.

It is also understood that an element or step “selected from the groupconsisting of” refers to one or more of the elements or steps in thelist that follows, including combinations of any two or more of thelisted elements or steps.

Unless indicated otherwise, the meaning of other terms will be clearfrom the context or will be understood to be the same as understood andused by persons skilled in the art.

Persons skilled in the art also understand that a nucleic acid sequence(nucleotide base sequence) as described herein provides the DNA, RNA,and complementary sequences thereof.

Recombinant expression vector elements (rEVEs) of the invention werefirst isolated from the regions of genomic DNA flanking either side ofthe site of integration of an expression vector present in cells of atransfected DUXB11 CHO cell line that expressed exceptionally highlevels of anti-IL-12 antibody molecules from genes present on theexpression vector. Cloning and sequencing of these flanking regions ofDUXB11 CHO genomic DNA revealed the sequence of SEQ ID NO:1 and of SEQID NO:2. The 2329 base sequence of SEQ ID NO:1 is also referred hereinas “ARM1”, and the 2422 base sequence of SEQ ID NO:2 is referred to as“ARM2”. The flanking ARM1 DNA sequence was shown to be located upstreamof the direction of transcription of the heavy chain gene of theintegrated expression vector, and the flanking ARM2 DNA sequence wasshown to be located downstream of the direction of transcription of thelight chain gene of the integrated expression vector (see, Examplessection, below).

ARM1- and ARM2-containing DNA molecules were inserted alone and incombination in expression vectors to determine whether their presencecould affect the level of expression of various recombinant proteins inhost cells containing the expression vectors. Both ARM1- andARM2-containing DNA provide enhanced levels of expression of recombinantproteins in host cells compared to the levels of production obtained inthe absence of these sequence (see, Examples, below). Accordingly, ARM1-and ARM2-containing nucleic acid molecules are examples of recombinantexpression vector elements (rEVEs). Typically, the enhanced level ofproduction obtained with an ARM1 polynucleotide is somewhat lower thanthat obtained with ARM2 polynucleotide or with a combination of ARM1 andARM2 polynucleotides.

In all instances where either or both of ARM1 and ARM2 have beenemployed in the construction of expression vectors for the production ofa recombinant protein of interest according to the invention, the levelof expression of the protein was greater using the ARM1 and/or ARM2expression enhancer elements. Increased comparative expression levelsfor the recombinant protein product of greater than 2-fold, greater than3-fold, greater than 4-fold, greater than 5-fold, greater than 6-fold,greater than 9-fold, greater than 10-fold, greater than 13-fold, greaterthan 16-fold, and greater than 18-fold over control expression systemshaving no ARM enhancers were directly observed. (See Examples, below.)Moreover, the high level of expression obtained using expression vectorsbearing ARM1 or ARM2, or both ARM1 and ARM2, is stably maintained overtime in the transfected host cells.

ARM1 and ARM2 sequences possess regions that are relatively rich insequences for matrix/scaffold attachment regions (MARS/SARs; see, e.g.,Michalowski et al., Biochemistry, 38: 12795-12804 (1999); Tikhonov etal., The Plant Cell, 12: 2490-264 (2000) U.S. Pat. No. 7,129,062; Bodeet al., Int. Rev. Cytol., 162A: 389-454 (1995); Bode et al., Crit. Rev.Eukaryotic Gene Expression, 6: 115-138 (1996)). In the case of ARM1, MARsequence motifs are particularly concentrated in the 3′ terminal regionof the sequence, whereas in the case of ARM2, clusters of MAR motifsappear in both the 5′ and 3′ terminal regions of the sequence with fewerMAR motifs situated in the middle region of the sequence. Deletionanalysis indicates that an approximately 1260 base pair 3′ terminalregion of ARM2 nucleic acid contains sequences that are critical forARM2 enhanced protein expression activity (see, Example 6, below).

The rEVE molecules of the invention include those nucleic acid moleculesthat comprise expression enhancing portions or fragments of the ARM1 andARM2 sequences. Such expression enhancing portions of the ARM1 and ARM2sequences are those that, when present on the same expression vector asa gene encoding a recombinant protein of interest, enhance the level ofexpression of the recombinant protein in a host cell containing theexpression vector as compared to that level of expression obtained inthe absence of such portions or fragments of ARM1 or ARM2 sequences.

A rEVE described herein may be used alone or in combination with anyother regulatory element or expression enhancing system for elevatingthe level of production of a desired recombinant protein(s) in a hostcell. Such other sequences and systems may include, without limitation,the use of regulated promoters to control or optimize transcription ofthe gene(s) encoding a recombinant protein(s) of interest, the use asignal sequence that directs secretion of recombinant protein(s) fromthe host cell, and the use of gene amplification methods, such as adihydrofolate reductase (DHFR)-methotrexate (MTX) amplification protocolor a glutamine synthetase protocol. In a DHFR-MTX amplificationprocedure, host cells carrying an expression vector that comprises agene for DHFR are selected for resistance to increasing concentrationsof methotrexate (see, e.g., Kaufman, R. J., In Genetic Engineering:Principles and methods (ed. J. K. Setlow), volume 9, page 155 (PlenumPublishing, New York, 1987)). Typically, as the level of resistance tomethotrexate increases, there is an accompanying amplification of thecopy number of the recombinant gene(s) along with a correspondingamplification (elevation) in the level of recombinant protein expressionfrom such amplified gene(s).

The nucleic acid sequences of SEQ ID NO:1 and SEQ ID NO:2 wereidentified from DNA molecules obtained from a DUXB11 CHO cell line. Anyof a variety of methods may be used to produce nucleic acid moleculescomprising a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, orportions of either of these sequences. Such methods include, withoutlimitation, cloning one or more rEVE DNA molecules comprising suchsequences out of the genomic DNA of DUXB11 CHO cells, solid-phasenucleic acid synthesis protocols to produce a polynucleotide comprisingsuch sequences, recombinant nucleic acid techniques, polymerase chainreaction (PCR) protocols, automated nucleic acid synthesizers, andcombinations thereof. A rEVE polynulecotide molecule described hereinmay also be purchased from commercial suppliers of custom designednucleic acid molecules. The method by which a rEVE is produced orsynthesized is not limiting, and the skilled practitioner can decidewhich method or combination of methods is most appropriate for producinga particular rEVE described herein.

A rEVE described herein may be inserted into an expression vector toenhance expression of any of a variety of proteins (including peptides,polypeptides, and oligomeric proteins) from an expression vector in ahost cell. Provided a nucleotide coding sequence(s) is known oravailable on a nucleic acid (DNA or RNA) molecule, the codingsequence(s) or the entire functional gene for a protein of interest maybe inserted into an expression vector using any of a variety of methodsavailable in the art. Coding sequences are operably linked to a promoterthat will function in the type of host cell for which expression isdesired. Additional transcriptional and translational sequences may alsobe engineered into the vector for the proper expression of the desiredprotein(s) in the host cell.

The expression of any of a vast variety of proteins (including peptides,polypeptides, oligomeric proteins) from an expression vector in anappropriate host cell may be enhanced by one or more rEVEs describedherein, including, but not limited to, soluble proteins, membraneproteins, structural proteins (i.e., proteins that provide structure orsupport to cells, tissues, or organs), ribosomal proteins, enzymes,zymogens, various antibody molecules (including, but not limited to,antibodies to TNF-α, such as adalimumab; antibodies to selectins;antibodies to immunoregulatory proteins, such as antibodies to IL-13;dual variable domain immunoglobulin molecules, such as DVD-IG™ dualvariable domain immunoglobulin molecules; antibodies to cell surfacereceptors), cell surface receptor proteins, transcription regulatoryproteins, translation regulatory proteins, chromatin proteins, hormones,cell cycle regulatory proteins, G proteins, neuroactive peptides,immunoregulatory proteins (e.g., interleukins, cytokines, lymphokines),blood component proteins, ion gate proteins, heat shock proteins,dihydrofolate reductase, an antibiotic resistance protein, a functionalfragment of any of the preceding proteins, an epitope-containingfragment of any of the preceding proteins, and combinations thereof.

A recombinant protein of interest may be produced by transcribing andtranslating the gene or genes that encode the recombinant protein andare present on an expression vector molecule described herein. Suchtranscription and translation of the gene or genes on an expressionvector, including those described herein, may be carried out using acell-free transcription/translation system or using an appropriate hostcell that contains the expression vector molecule and that has theproper cellular transcription and translation machinery necessary toproduce the recombinant protein of interest from the gene or genespresent on the expression vector molecule.

Matrix attachment regions (MARs), also referred to as scaffoldattachment regions (SARs), were originally identified as fragments ofchromosomal DNA that bind to preparations of nuclear proteins thatappear to form a scaffold or matrix in the eukaryotic nucleus. MARs arebelieved to reside in loops of chromatin that attach to the nuclearmatrix and to define or delimit individual structural units ofchromatin. MARs have been associated with some enhancer sequences and/orlinked to a number of processes that may occur in chromatin, includingtranscription, transgene expression, transgene rearrangement,recombination, replication, and stabilization of transfections (see,e.g., Michalowski et al., Biochemistry, 38: 12795-12804 (1999); Zhong etal., Proc. Natl. Acad. Sci. USA, 96(21): 11970-11975 (1999); Tikhonov etal., The Plant Cell, 12: 249-264 (2000); Zhou et al., Gene, 277: 139-144(2001); Sumer et al., Genome Research, 13: 1737-1743 (2003); U.S. Pat.No. 7,129,062; Bode et al., Int. Rev. Cytol., 162A: 389-454 (1995); Bodeet al., Crit. Rev. Eukaryotic Gene Expression, 6: 115-138 (1996)). ARM1(SEQ ID NO:1) and ARM2 (SEQ ID NO:2) possess a variety of MAR elements,which appear to be located predominantly in clusters in the 3′ and/or 5′terminal regions. ARM1 contains a cluster of MAR elements in the 3′terminal region. In the case of ARM2, MAR elements can be foundthroughout the length of this rEVE sequence with clusters of MAR motifslocated in both the 5′ and the 3′ terminal regions. Clearly, vectors andother nucleic acid molecules carrying ARM1 and ARM2 sequences areconvenient sources of MAR elements and clusters of MAR elements.Accordingly, ARM1, ARM2, and MAR-containing portions thereof may be usedto increase the number of MAR elements in a nucleic acid molecule ofinterest using any of the various methods available in the art forrecombining or otherwise transferring nucleic acid sequence informationto a nucleic acid molecule.

Nucleic acid molecules comprising a sequence of a rEVE described hereinor a portion thereof may also be used in a variety of procedures knownin the art to manipulate, identify, produce, or amplify rEVE sequencesin other nucleic acid molecules. Such procedures may include, withoutlimitation, use of a rEVE or portion thereof as a probe in any of thevarious nucleic acid hybridization methods known in the art or as aprimer in any of the various polymerase chain reaction (PCR) proceduresknown in the art.

A rEVE polynucleotide molecule described herein may be used in methodsto produce a recombinant protein of interest. Preferably, such a methodof producing a recombinant protein of interest comprises a recombinantexpression vector as described herein that comprises one or morerecombinant genes encoding the recombinant protein of interest and atleast one rEVE polynucleotide molecule described herein, andtranscribing and translating the one or more recombinant genes presenton the recombinant expression vector to produce the recombinant proteinsof interest. The transcription and translation of the one or morerecombinant genes from the expression vector is preferably carried outin a host cell that comprises the expression vector and is culturedunder conditions promoting expression of the recombinant protein ofinterest. A rEVE polynucleotide molecule may be used to enhance thelevel of expression of one or more recombinant proteins of interest froman expression vector in a host cell, whether transiently expressed(e.g., in a transfected COS or HEK 293 host cell) or stably expressed(e.g., in a transfected CHO host cell). Transcription and translation ofone or more recombinant genes from an expression vector that carries arEVE as described herein may also be carried out using an in vitrocell-free transcription/translation system available in the art.

A rEVE polynucleotide molecule described herein may be used in preparinga host cell that stably (as opposed to transiently) expresses elevatedlevels of a recombinant protein when the level of expression has beenamplified (elevated) using a DHFR-methotrexate amplification procedure.Such host cells are methotrexate-resistant (MTX-resistant) cells thatwill continue to express elevated levels of a recombinant protein notonly when grown in the presence of methotrexate, but also when grown inthe absence of methotrexate, i.e., when grown in the absence of theselective pressure for elevated expression provided by the presence ofmethotrexate. In a preferred embodiment, such a method to produce amethotrexate-resistant host cell comprises the steps of:

-   -   inserting into host cells an expression vector comprising:        -   a recombinant gene coding for a recombinant protein of            interest,        -   a rEVE or expression enhancing portion thereof, and        -   a dihydrofolate reductase (DHFR) gene,    -   growing the host cells in the presence of methotrexate to select        for a methotrexate-resistant host cell that expresses the        recombinant protein of interest, and    -   isolating a methotrexate-resistant host cell;        wherein the isolated methotrexate-resistant host cell expresses        the recombinant protein of interest at a level that is higher        than that of a methotrexate-sensitive host cell, and wherein        said methotrexate-resistant host cell stably expresses an        elevated level of the recombinant protein when grown in the        presence or in the absence of methotrexate. Preferably, the rEVE        comprises SEQ ID NO:1, SEQ ID NO:2, or an expression enhancing        portion thereof. More preferably, the rEVE comprises SEQ ID        NO:2.

The rEVE polynucleotide molecules described herein may also be used in amethod of improving the ability of a population of host cells thatexpress a recombinant protein to adapt to growth in the presence ofmethotrexate. In a preferred embodiment, such a method comprises:

-   -   inserting into host cells an expression vector comprising:        -   a recombinant gene encoding a protein of interest,        -   a recombinant expression vector element (rEVE)            polynucleotide molecule comprising a sequence selected from            the group consisting of SEQ ID NO:1, SEQ ID NO:2, a portion            SEQ ID NO:1, a portion of SEQ ID NO:2, and combinations            thereof, and        -   a dihydrofolate reductase (DHFR) gene;            wherein a population of the host cells containing the            expression vector has a higher survivability and/or higher            growth rate when grown in the presence of methotrexate            compared to a population of host cells carrying the            expression vector lacking said rEVE polynucleotide molecule.

A rEVE polynucleotide molecule described herein may also be employed toenhance the amplification (elevation) of expression of a recombinantprotein in host cells using a DHFR-methotrexate amplification procedure.In a preferred embodiment, such a method comprises:

-   -   inserting into host cells an expression vector comprising:        -   a recombinant gene coding for a recombinant protein of            interest,        -   a rEVE polynucleotide molecule comprising a sequence            selected from the group consisting of SEQ ID NO:1, SEQ ID            NO:2, a portion of SEQ ID NO:1, a portion of SEQ ID NO:2,            and combinations thereof, and        -   a dihydrofolate reductase (DHFR) gene,    -   growing the host cells in the presence of methotrexate to select        for a methotrexate-resistant host cell that expresses the        recombinant protein of interest, and    -   isolating the methotrexate-resistant host cell,        wherein the isolated methotrexate-resistant host cell expresses        the recombinant protein of interest in the presence of        methotrexate at a level that is higher than that of a        methotrexate-resistant host cell containing an expression vector        lacking said rEVE polynucleotide molecule.

In methods described herein that employ a methotrexate selection foramplified expression of a desired recombinant protein(s), methotrexatemay be used in the range of 20 nM to 500 nM. However, advantageouslypersons skilled in the art recognize that lower and higher methotrexateconcentrations, such as 5 nM to 10 μM, may also be successfully employedin methotrexate selections to amplify expression (see, e.g., Kaufman, R.J., Methods in Enzymology, Vol. 185: 537-566 (1990)).

The invention also provides a method of lowering, substantiallysuppressing, or essentially silencing expression of a recombinantprotein from an expression vector. Such a method may employ anexpression vector that comprises one or more fragments of a rEVEsequence described herein that provide lower levels of expression of aparticular recombinant gene product than provided using a full-lengthrEVE sequence. Such expression-lowering or expression-suppressingrEVE-derived sequences include a truncated variant of the sequence ofARM2 having the base sequence of bases 1-1086 of SEQ ID NO:2 or bases1-461 of SEQ ID NO:2. Clearly, the presence of the 3′ terminal sequenceregion deleted from these two variants is highly desirable forrEVE-mediated enhanced expression of recombinant proteins. A nucleicacid molecule having the ARM2 truncated sequence of bases 1-461 of SEQID NO:2 is particularly useful in suppressing or substantially silencingexpression of a recombinant protein from an expression vector moleculein a host cell. Such methods may find use in a number of situations,including, without limitation, when there is a concern that expressionof a recombinant protein may be toxic to the host cells or when somelevel of expression may present problems to purification or isolation ofthe desired protein, such as protein aggregation.

Thus, the sequence of bases 462-2422 of SEQ ID NO:2 and the sequence ofbases 1087-2422 of SEQ ID NO:2 are most preferred for maximal ARM2rEVE-mediated enhancement of expression of recombinant proteins.Accordingly, rEVE polynucleotides that are particularly useful forenhancing expression of recombinant proteins comprise the sequence ofbases 462-2422 of SEQ ID NO:2 and/or the sequence of bases 1087-2422 ofSEQ ID NO:2.

Additional embodiments and features of the invention will be apparentfrom the following non-limiting examples.

EXAMPLES Example 1 Cloning and Sequence Analysis of ARM1 and ARM2Recombinant Expression Vector Elements (rEVEs)

A production DUX B11 CHO cell line carrying a stably integratedexpression vector was studied to determine the possible basis for theexceptionally high level of expression of a recombinant anti-IL-12antibody encoded on the expression vector with which the host CHO cellswere transfected. In the study, the regions of genomic DNA flanking theintegrated expression vector were cloned and sequenced. Briefly, genomicDNA from the host cell was digested with XbaI, and fragments of anaverage size of approximately 5 kilobases (kb) were purified using aBioGel A-50 column basically as described by Reynaud et al. (J. Mol.Biol., 140: 481-504 (1980)). A library of the purified genomic fragmentswas prepared by cloning the fragments into the lambda (λ) DASH® cloningvector (Stratagene, La Jolla, Calif.). The library was amplified andpackaged using GIGAPACK® packaging extract (Stratagene) and transducedinto XL1-Blue MRA or XL1-Blue MRA(P2) Escherichia coli cells accordingto the manufacturer's protocols. The library was then screened withprobes for the coding sequence of the heavy chain variable region(V_(H)) of the anti-IL-12 antibody according to Stratagene protocols. A14.5 kb Xba1 fragment in λDASH clone#21 contained the insertion site forthe antibody expression vector along with flanking genomic sequences.These flanking genomic regions were designated ARM1 and ARM2. Thesequences of ARM1 and ARM2 regions were determined to be that of SEQ IDNO:1 and SEQ ID NO:2, respectively. With respect to the insertion siteof the antibody expression vector in the CHO cell genome, ARM1 islocated upstream of the insertion site and 5′ to the direction oftranscription of the vector gene for the antibody heavy chain, and ARM2is located downstream of the insertion site and 3′ to the direction oftranscription of the vector gene for the antibody light chain.

A BLAST analysis of SEQ ID NO:1 and SEQ ID NO:2 against a CHO expressedsequence tag (EST) data base failed to reveal any related codingsequences, suggesting that the ARM1 and ARM2 rEVE sequences do notcontain any CHO coding sequences. A BLAST analysis of the rEVE sequenceswas also conducted against the mouse genome sequence. No mouse genomicsequence was identified that was homologous to the ARM1 sequence.However, the ARM2 sequence appears to be related to a highly conservedarea on mouse chromosome 14 that contains no known coding sequences.

Using the Vector NTI sequence analysis program (Invitrogen), thesequences of ARM1 (SEQ ID NO:1) and ARM2 (SEQ ID NO:2) were alsoscreened for the presence of a sampling of various representative (i.e.,not all) nucleotide sequence motifs for matrix attachment region (MAR)elements (see, e.g., Michalowski et al., Biochemistry, 38: 12795-12804(1999)). The specific MAR DNA sequence motifs screened by the computerprogram are shown in Table 1 (complementary strand sequences notlisted).

TABLE 1Nucleotide Sequence Motifs for Representative Matrix Attachment RegionMAR Element MAR Sequence Motif* Screened A-BoxAAYAAAYAA (SEQ ID NO: 3) (2 mismatches allowed) T-BoxTTWTWTTWTT (SEQ ID NO: 4) (1 mismatch allowed) MRSTAWAWWWNNAWWRTAANNWWG (SEQ ID NO: 5) (2 mismatches allowed, but 3′terminus is G) or TAWAWWW(bases 1-7 of SEQ ID NO: 5) (first part of MRS, no mismatches) +AWWRTAANNWWG (bases 10-21 of SEQ ID NO: 5)(second part of MRS, 1 mismatch allowed, but 3′ terminusis G), where first and second MRS parts counted with upto 4 intervening bases (N₁₄) or up to 3 overlapping bases ARSWTTTATRTTTW (SEQ ID NO: 6) (1 mismatch allowed) BURAATATATTT (SEQ ID NO: 7) (1 mismatch allowed) CurvedAAAANNNNNNNAAAANNNNNNNAAAA (SEQ ID NO: 8) or TTTAAA (SEQ ID NO: 9)90% AT W₂₀ (SEQ ID NO: 10) (2 mismatches allowed) KinkedTANNNTGNNNCA (SEQ ID NO: 11) or TANNNCANNNTG (SEQ ID NO: 12)or TGNNNTANNNCA (SEQ ID NO: 13) or CANNNTANNNTG(SEQ ID NO: 14) (no mismatches allowed) Topoisomerase IIGTNWAYATTNATNNR (SEQ ID NO: 15) (2 mismatches allowed, but Binding Sitenot in positions 4-9, i.e., WAYATT (bases 4-9 ofSEQ ID NO: 15) conserved) TG-Rich CAAAACA (SEQ ID NO: 16) DNA UnwindingAATATT (SEQ ID NO: 17) or AATATATT (SEQ ID NO: 18) Motif *complementarystrand sequences not listed; abbreviations: Y = T or C; W = A or T; N =A, T, G, or C; R = G or A

As shown in Table 2, below, at least 41 MAR element sequences wereidentified in the ARM1 DNA sequence (SEQ ID NO:1) or its complementarystrand sequence, and at least 114 MAR element sequences were identifiedin the ARM2 DNA sequence (SEQ ID NO:2) or its complementary strandsequence.

TABLE 2 Location of MAR Sequences in ARM1 and ARM2 DNA SequencesLocation in ARM1 Sequence Location in ARM2 Sequence MAR Element (basesof SEQ ID NO: 1)* (bases of SEQ ID NO: 2)* A-Box 1756-1764, 1939-1947,52-60, 69-77, 79-87, 244-252 1975-1983, 2062-2070, (complementary),340-348 2072-2080, 2079-2087, (complementary), 446-454 2216-2224,2280-2288 (complementary), 481-489 (complementary), 484-492(complementary), 627-635, 631-639, 664-672 (complementary), 734-742,737-745, 741-749, 811-819 (complementary), 821-829, 833-841, 840-848,907-915 (complementary), 925-933 (complementary), 942-950(complementary), 1104-1112 (complementary), 1186-1194 (complementary),1186-1213 (cluster, complementary), 1262-1270 (complementary),1391-1399, 1409- 1417, 1413-1421, 1421-1429, 1441- 1449 (complementary),1531-1539 (complementary), 1711-1719 (complementary), 1715-1723(complementary), 1740-1748, 1810- 1818, 1817-1825, 1844-1852, 1848-1856, 1862-1870, 2036-2044 (complementary), 2040-2048 (complementary),2052-2060, 2100- 2108 (complementary), 2107-2115 (complementary),2113-2121, 2118- 2126, 2122-2130, 2191-2199 (complementary), 2261-2269(complementary), 2287-2295 T-Box 114-145 (cluster), 1740- 483-492,631-640, 734-743, 806-815, 1752 (cluster), 2000-2011 925-934, 1198-1207,1843-1852 (cluster), 2062-2087 (cluster) MRS 130-151, 1969-1989, 2054-77-97, 401-421, 752-772, 1197-1213 2074, 2266-2286 ARS 121-131,2062-2072, 2079- 1443-1453 2089, 2126-2136, 2257- 2267 BUR 1877-1885,2005-2013, 77-85, 255-263, 723-731, 1412-1420 2063-2071, 2098-2106(complementary), 2281- 2289 Curved 106-131, 2011-2016, 437-442, 466-471,530-535, 616-621, 2293-2298 627-632, 2151-2156 90% AT 106-125, 126-14564-83, 64-90 (cluster), 243-263 (cluster), 400-420, 613-645 (cluster),698-717, 722-774 (cluster), 805-845 (cluster), 1185-1214 (cluster),1407- 1431 (cluster), 2104-2136 (cluster) Kinked 860-871, 1761-1772439-450, 671-682, 1124-1135, 1503- 1514, 1967-1978, 2020-2031, 2058-2069 Topoisomerase II 598-612, 1365-1382, 2042- 720-734, 819-833(complementary), Binding Site 2056 (complementary) 1251-1265, 1407-1421(complementary), 1415-1429 (complementary), 1418-1432, 1434- 1448(complementary), 1758-1775, 1981-1995, 2202-2216 (complementary) TG-Rich252-258 (complementary) 928-936, 1842-1848 (complementary) DNA Unwinding1880-1885, 2021-2026, 284-289, 415-420, 788-793, 789-794, Motif2064-2069, 2066-2071 825-830, 826-831, 909-914, 1121- 1126, 1421-1426,1440-1446, 2208- 2213, 2209-2214 *ARM1 sequence (SEQ ID NO: 1) or ARM2sequence (SEQ ID NO: 2); “complementary” = complementary strandsequence; “cluster” = more than one copy of indicated MAR element inspecified sequence

The results of the MAR sequence analysis (which included complementarystrand analysis) of ARM1 and ARM2 in Table 2 indicate that the variousMARs are clustered predominantly in the 3′ terminal portion of ARM1 (SEQID NO:1) and in both the 5′ portion and 3′ portion of ARM2 (SEQ IDNO:2). ARM1 has fewer of these MAR sites than ARM2. Moreover, themajority of the MAR sequences in ARM1 are in the 3′ region of thesequence, whereas both the 5′ and the 3′ regions of ARM2 are populatedby MAR sequences.

Example 2 General Protocols for Studies of the Affect of ARM1 and ARM2rEVEs on Levels of Expression of Recombinant Proteins Cells forExpression Studies

The DUXB11 CHO cell line is a CHO cell line that is deficient inexpression of dihydrofolate reductase (DHFR⁻) (see, Urlaub, G. andChasin, L. A., Proc. Natl. Acad. Sci. U.S.A., 77: 4216-4220 (1980)). TheDHFR⁻ phenotype of this cell line permits the use of a standarddihydrofolate reductase-methotrexate system (DHFR-MTX) protocol toamplify the copy number of an expression vector that has beentransfected into these cells (see, e.g., Kaufman, R. J., In GeneticEngineering: Principles and Methods (ed. J. K. Setlow), volume 9, page155 (Plenum Publishing, New York, 1987)).

Expression Vectors Containing ARM1 and ARM2 rEVE Sequences

FIG. 1 provides a schematic diagram of plasmid expression vector pA205.FIG. 2 provides a schematic diagram of plasmid expression vectorpA205Genomic in which the genes encoding the heavy and light chains foradalimumab are flanked by ARM1 and ARM2 such that an ARM1 sequence islocated upstream of the gene for the heavy chain of adalimumab and anARM2 sequence is located downstream of the gene for the light chain ofadalimumab. FIG. 3 provides a schematic diagram of plasmid expressionvector pA205A1 in which an ARM1 sequence is located upstream of the genefor the heavy chain of adalimumab. FIG. 4 provides a schematic diagramof plasmid expression vector pA205A2 in which an ARM2 sequence islocated downstream of the gene for the light chain of adalimumab.

FIG. 5 provides a schematic diagram of plasmid expression vectorpCHOEL246GGhum, which contains the genes for the heavy and light chainsof a human anti-EL-selectin antibody. FIG. 6 provides a schematicdiagram of plasmid expression vector pA-Gen-EL-Selectin in which thegenes encoding the heavy and light chains for the anti-EL-selectinantibody are flanked by ARM1 and ARM2 sequences such that an ARM1sequence is located upstream of the gene for the antibody heavy chainand an ARM2 sequence is located downstream of the gene for the antibodylight chain.

FIG. 7 provides a schematic diagram of plasmid expression vectorpA205-eGFP, which contains the gene for enhanced Green FluorescentProtein (eGFP). FIG. 8 provides a schematic diagram of plasmidexpression vector pA205G-eGFP in which the gene encoding the enhancedGreen Fluorescent Protein is flanked by ARM1 and ARM2 sequences suchthat the ARM1 sequence is located upstream of the gene for eGFP and theARM2 sequence is located downstream of the eGFP gene.

FIG. 9 provides a schematic diagram of plasmid expression vectorpA205A2-Spe-trunc, which is essentially identical to expression vectorpA205A2 shown in FIG. 4, except that the ARM2 sequence has been replacedwith a truncated ARM2 variant, “A2 (bp 1-1086)”, which has the nucleicacid base sequence of bases 1-1086 of SEQ ID NO:2 as the result ofdigestion of ARM2 DNA with restriction endonuclease SpeI. FIG. 10provides a schematic diagram of plasmid expression vectorpA205A2-Swa-trunc, which is essentially identical to expression vectorpA205A2 shown in FIG. 4, except that the ARM2 sequence has been replacedwith a truncated ARM2 variant, “A2 (bp 1-461)”, which has the nucleicacid base sequence of bases 1-461 of SEQ ID NO:2 as the result ofdigestion of ARM2 DNA with restriction endonuclease Swa1.

Culture Media

αMEM is minimal essential medium, α medium (GIBCO®, Invitrogen,Carlsbad, Calif.).

Growth of Cells and Transfections

Three 10 cm tissue culture plates per transfection were seeded with1×10⁶ B3.2 parental DUXB11 CHO cells each in 10 ml of αMEM supplementedwith 5% dialyzed FBS and H/T (GIBCO). These cells were transfectedeighteen hours (18 h) later in accordance with the calcium phosphatetransfection protocol described in Current Protocols in MolecularBiology, (Ausubel, F. V., Brent, R., Moore, D. M., Kingston, R. E.,Seidman, J. G., Smith, J. A., and K. Struhl eds.), (Wiley Interscience,New York, 1990)) with several modifications as indicated, below. Growthmedium was removed from culture plates by aspiration, and 9 ml of Ham'sF12 medium (Invitrogen) was added to each plate. The plates wereincubated at 37° C. for two hours prior to transfection.

Calcium Phosphate Transfection Protocol

Seventy-five micrograms (75 μg) of DNA were dissolved in 1.35 ml waterin a 50 ml conical tube. One hundred and fifty microliters (150 μl) of2.5 M CaCl₂ were added, and this DNA-calcium mixture was added dropwiseto 1.5 ml of 2× HeBES (HEPES buffered saline) in a 50 ml conical tube.The HeBES was bubbled with a pipettor while adding the DNA-calciummixture with a Pasteur pipette. The mixture was mixed by vortex for 5seconds and incubated at room temperature for 20 minutes. One milliliter(1 ml) of the DNA-calcium mixture was distributed evenly over eachculture dish of adherent cells, grown and prepared for transfection inF12 medium as described above, and the cultures then incubated at 37° C.for four hours. After incubation, the plates were aspirated, and 2 ml of10% dimethylsulfoxide (DMSO) in F12 medium was added to each plate (DMSOshock treatment). The DMSO shock continued for one minute after whichthe DMSO was diluted by the addition of 5 ml of PBS (phosphate bufferedsaline) to each plate. The plates were aspirated and washed two moretimes in PBS. Ten (10) ml of αMEM/5% FBS/HT was added, and the plateswere incubated at 37° C. overnight.

Seeding of Transfected Cells into 96-Well Plates and Methotrexate (MTX)Amplification

The next day, the cells were seeded into 96-well plates as follows: Thecells from all of the 10 cm plates were harvested by trypsin digestionand resuspended at a density of 2000 cells/ml in αMEM/5% FBS. Ninety-six96-well plates were seeded at 10 ml/plate, 100 μl/well. The medium waschanged on the 96-well plates one week later, two weeks later, and againfive days after that. The medium αMEM/5% FBS used was selective forcells expressing DHFR.

Two days after the last medium change, the culture supernatants werediluted 1:40 and tested using an ELISA specific for human IgG gammachain to detect expression of adalimumab or anti-EL-selectin antibody.The clones which gave the highest ELISA signal were transferred fromtheir 96-well plates into 12-well plates in 2.0 ml/well of αMEM/5% FBS.When confluent (approximately 2-5 days later), these were assayed again,and clones were split into the same medium+20 nM MTX for amplification.Cells were initially selected in αMEM/5% FBS and 20 nM MTX in a 12-welltissue culture plate. These cells, after initial selection at this MTXlevel were passed a minimum of two more times in growth mediumcontaining 20 nM MTX over an average period of eighteen days. The celllines were amplified then to 100 nM MTX. During this period of culture,the adalimumab (human anti-TNF-α antibody) or anti-EL-Selectinproductivity of the cultures increased. After selection at 100 nM MTXlevel, the lines were passed a minimum of two more times in growthmedium containing 100 nM MTX over an average period of eighteen (18)days. Where indicated, the cell lines were further amplified to 500 nMMTX. After selection at 500 nM MTX level, the lines were passed aminimum of two more times in growth medium containing 500 nM MTX over anaverage period of eighteen (18) days.

Cultures of transfected CHO cells carrying expression vectors comprisingan ARM1 nucleic acid sequence (SEQ ID NO:1), an ARM2 nucleic acidsequence (SEQ ID NO:2), or a combination of ARM1 and ARM2 sequences,adapt better, i.e., have higher survivability and/or higher growthrates, in the presence of methotrexate compared to CHO cells transfectedwith the same expression vectors lacking these rEVE sequences.

Example 3 Effect of ARM1 and ARM2 Sequences on Levels of Expression ofAnti-TNF-α in Transfected CHO Cells

In this study, the effect of ARM1 and ARM2 sequences on the expressionof a human anti-TNF-α antibody (adalimumab) in stably transfected CHOcells was examined. The levels of expression of adalimumab were comparedin CHO cells stably transfected with expression vector pA205 (no ARMsequences), pA205Genomic (containing both ARM1 and ARM2), pA205A1(containing ARM1), or pA205A2 (containing ARM2). Table 3 shows theaverage level of production of adalimumab in adherent cultures of thetop three producing clones from each vector transfection at theindicated amplification level (concentration of methotrexate, “MTX”).

TABLE 3 Level of Adalimumab Expression in Stably Transfected CHO CellsVector 0 nM MTX 20 nM MTX 100 nM MTX pA205 1.57 μg/ml  4.20 μg/ml 5.53μg/ml pA205Genomic 4.60 μg/ml 14.80 μg/ml 21.03 μg/ml  pA205A1 3.13μg/ml  9.90 μg/ml 17.3 μg/ml pA205A2 4.53 μg/ml 24.23 μg/ml 20.8 μg/ml

The data in Table 3 indicate that ARM1 and ARM2 sequences, alone or incombination, enhanced the level of expression of adalimumab compared tothat seen in cells transfected with a vector lacking ARM sequences(pA205). Overall, ARM2 alone (p205A2) provided the greatest level ofenhanced expression relative to all other transfectants, whereas ARM1alone (p205A1) provided the lowest of enhanced levels of expression. Thedata show that an isolated nucleic acid molecule that has the ARM1sequence (SEQ ID NO:1) or ARM2 sequence (SEQ ID NO:2) is arepresentative recombinant expression vector element (rEVE), which wheninserted into an expression vector can enhance expression of arecombinant protein encoded by and expressed from the expression vector.

The levels of expression of adalimumab by individual clones of theabove-mentioned vector transfections were also compared after 1 week and4 weeks in suspension cultures in the absence of methotrexateamplification. Results are shown in Table 4.

TABLE 4 Level of Adalimumab Expression in Suspension Cultures ofIndividual Clones of Stably Transfected CHO Cells after Week 1 and 4 inthe Absence of Methotrexate Week 1 Week 4 Vector Clone # (μg/ml) (μg/ml)pA205 19-3  10.8 8.1 pA205Genomic 7-5 52.6 46.0 14-6  19.9 16.8 pA205A11-4 15.5 6.9 4-2 27.8 15.3 6-3 76.8 43.6 3-7 17.3 12.6 pA205A2 13-1 97.7 107.8 9-7 63.7 64.9 7-2 42.3 60.2 5-3 180.4 153.0

The data in Table 4 show that incorporating ARM2 into pA205 without ARM1(pA205A2) increases the stability of enhanced adalimumab expressionlevels over time. In particular, with both ARM1 and ARM2 present(pA205Genomic) or ARM1 alone (pA205A1), an enhanced level of expressionof adalimumab was initially observed in cultures, but that level couldquickly fall over a matter of weeks to much lower levels (see, Table 4).In contrast, in cultures of cells expressing adalimumab in the presenceof ARM2 alone (pA205A2), the enhanced level of expression of adalimumabwas stably maintained in three out of four cultures over the course ofthe 4 week period. Thus, ARM2 can stably maintain a significantelevation in the level of expression of adalimumab in suspensioncultures of transfected CHO cells.

Example 4 Effect of ARM1 and ARM2 Sequences on the Level of Expressionof Anti-EL-Selectin Antibody in Transfected CHO Cells

In this study, the effect of ARM1 and ARM2 sequences on the expressionof a human anti-EL-selectin antibody (which binds E- and L-selectins) instably transfected CHO cells was examined. The levels of expression ofthis anti-selectin antibody were compared in CHO cells stablytransfected with expression vector pCHOEL246GGhum (no ARM sequences) orwith expression vector pA-Gen-EL-Selectin (containing both ARM1 andARM2). Table 5 shows the average level of production of theanti-selectin antibody in adherent cultures of the top three producingclones from each vector transfection at the indicated amplificationlevel (concentration of methotrexate, “MTX”).

TABLE 5 Level of Anti-EL-Selectin Antibody Expression in StablyTransfected CHO Cells Vector 0 nM MTX 20 nM MTX 100 nM MTX 500 nM MTXpCHOEL246GGhum 7.93 μg/ml 15.47 μg/ml 17.27 μg/ml 22.30 μg/mlpA-Gen-EL-Selectin 6.27 μg/ml 19.87 μg/ml 26.63 μg/ml 44.13 μg/ml

The data in Table 5 show that ARM1 and ARM2 sequences(pAGen-EL-Selectin) were effective in elevating the level of expressionof anti-EL-selectin antibody in methotrexate-amplified cultures ofstably transfected CHO cells.

Example 5 Effect of ARM1 and ARM2 Sequences on Levels of Expression ofEnhanced Green Fluorescent Protein (eGFP) in Transfected Cho Cells

Constructs containing a gene encoding enhanced green fluorescent protein(eGFP) were transfected into CHO cells as described above with theexception that the cells were co-transfected with pcDNA3.1-hygro toprovide a selectable marker, i.e., hygromycin resistance, for stabletransfectants. Expression vector peGFP provides a positive control forexpression of eGFP under the transcriptional control of a CMV promoter.Plasmid pA205-eGFP contains the eGFP gene on the pA205 expression vectorwithout ARM1 or ARM2 sequences (FIG. 7). Plasmid pA205Gen-eGFP (FIG. 8)contains both ARM1 and ARM2 flanking the gene encoding eGFP. There is noDHFR selection gene in either of these plasmids. Accordingly, noDHFR-methotrexate amplification steps were taken. Transfectants wereselected with hygromycin at a concentration of 400 μg/ml for 2 weeks,splitting when necessary, and then sorted by FACS to determineexpression levels. The cells were originally sorted into pools for eachtype of vector, and expressing cells were grown for an additional twoweeks, then resorted.

TABLE 6 Level of eGFP Expression in Stably Transfected CHO Cells VectorMean Fluorescence Units peGFP (positive control) 239.03 pA205-eGFP 17.36pA205Gen-EGFP 104.86

The data in Table 6 indicate that ARM1 and ARM2 sequences were effectiveat significantly enhancing the level of expression of eGFP intransfected CHO cells compared to the level of expression in cellstransfected with the same eGFP expression vector, but lacking ARMsequences (pA205-eGFP).

Example 6 Effect of Deletion Mutants of ARM2 on Levels of Expression ofAdalimumab in Transfected CHO Cells

The 3′ terminal region of the ARM2 sequence (SEQ ID NO:2) was truncatedin pA205A2 vector to either the SpeI cleavage site (at base 1086 of SEQID NO:2) or the SwaI cleavage site (at base 461 of SEQ ID NO: 2) tocreate, respectively, pA205A2-Spe-trunc (containing the sequence ofbases 1-1086 of SEQ ID NO:2) and pA205A2-Swa-trunc (containing thesequence of bases 1-461 of SEQ ID NO:2). The resulting plasmids weretransfected into CHO cells as described above and amplified to 20 nMMTX. Table 7 shows the average level of expression in adherent culturesof the top three producing clones from each transfection at eachamplification level.

TABLE 7 Level of Adalimumab Expression in Stably Transfected CHO CellsVector 0 nM MTX 20 nM MTX pA205 1.80 μg/ml 1.57 μg/ml pA205A2 6.10 μg/ml15.73 μg/ml  pA205A2-Spe-trunc 3.57 μg/ml 3.00 μg/ml pA205A2-Swa-trunc0.83 μg/ml 0.90 μg/ml

The data in Table 7 show that CHO cells transfected with expressionvector pA205A2-Spe-trunc, which contains the 5′ terminal 1086 base pairsof the ARM2 DNA, produced adalimumab at a lower level of enhancedexpression compared to CHO cells transfected with the pA205A2 vector.Surprisingly, cells transfected with expression vectorpA205A2-Swa-trunc, which contains the 5′ terminal 461 base pairs of theARM2 DNA, produced adalimumab at even lower levels of expression thancells transfected with pA205, which contains no ARM sequence. The datain Table 7 indicate that the regions deleted from the 3′ terminus of theARM2 DNA are of special interest for enhanced expression of recombinantproteins in host cells. In addition, the presence of a relatively small,i.e., 461 base pair, 5′ terminal fragment of ARM2 DNA, alone, actuallyappears to reduce expression of recombinant gene product in transfectedhost cells and may be particularly useful in substantially lowering orsilencing expression of a recombinant gene product. Such an applicationmay be desired when expression of a gene product would be toxic orotherwise undesired in a host cell, e.g., under certain cultureconditions.

Example 7 Expression Levels of Individual Transfectants from thePreceding Studies

The tables shown below provide the level of expression of recombinantproteins produced by individual transfected clones generated in thepreceding studies.

TABLE 8 Levels of Adalimumab Expression in CHO Cells Transfected withpA205 pA205 0 nM 20 nM 100 nM 500 nM clone# MTX MTX MTX MTX 1 3.9 10.415.4 14.1 2 2.8 6.2 12.9 8.2 3 2.50 4.2 10.1 2.9 4 2.1 3.8 4.2 1.5 5 1.72.7 2.9 0.3 6 1.4 2.2 2.6 7 1.4 1.7 2 8 1.3 1.6 1.7 9 1.3 1.6 1.4 10 1.21.6 1.4 11 1.10 1.4 1 12 1.10 1.3 0.8 13 1 1.3 0.5 14 1.00 1.3 0 15 0.91.2 16 0.90 1.2 17 0.9 1.1 18 0.9 1.1 19 0.9 1.01 20 0.9 0.9 21 0.80 0.822 0.8 0.8 23 0.8 0.7 24 0.70 0.7 25 0.70 0.6 26 0.7 0.6 27 0.7 0.3 280.5 0.1 29 0.50 0.09 30 0.50 0.08 31 0.5 0.07 32 0.5 0 33 0.5 0 34 0.4 035 0.4 0 36 0.40 0 37 0.40 0 38 0.40 0 39 0.40 0 40 0.4 0 41 0.4 0 420.3 0 43 0.3 44 0.30 45 0.3 46 0.3 47 0.2 48 0.20 49 0.2 50 0.2 51 0.152 0.1 53 0.1 54 0.1 55 0.10 56 0.10 57 0.10 58 0.1 59 0.08 60 0.05 610.05 62 0.03 63 0.03 64 0.03 65 0.02 66 0.01 67 0.01 68 0.01 69 0.01 700.01 71 0.009 72 0.007 73 0.007 74 0.007 75 0.006 76 0 77 0 78 0

TABLE 9 Levels of Adalimumab Expression in CHO Cells Transfected withpA205Genomic pA205Genomic 0 nM 20 nM 100 nM 500 nM clone# MTX MTX MTXMTX 1 9.3 19.1 61.0 40.6 2 6.9 16.5 59.7 40.2 3 6.5 15.1 52.0 28.6 45.70 13.6 36.0 23.9 5 5.6 13.2 25.9 6 4.6 10.7 20.7 7 4.3 10.3 19.6 84.1 9.8 17.6 9 4.10 9.7 16.3 10 4.00 9.5 15.6 11 3.9 8.9 11.4 12 3.6 7.69.3 13 3.5 6.9 8.1 14 3.4 6.4 8 15 3.3 6 5.9 16 2.9 4.5 0.1 17 2.9 3.10.04 18 2.70 2.5 19 2.60 2.4 20 2.5 1.6 21 2.4 1.4 22 2.2 1.1 23 2.20.05 24 2.2 0 25 2.20 0 26 2 27 2 28 1.90 29 1.90 30 1.8 31 1.80 32 1.733 1.7 34 1.6 35 1.6 36 1.6 37 1.50 38 1.4 39 1.4 40 1.40 41 1.3 42 1.343 1.30 44 1.30 45 1.2 46 1.1 47 1.1 48 1.1 49 1 50 1.00 51 0.80 52 0.753 0.7 54 0.7 55 0.6 56 0.60 57 0.5 58 0.5 59 0.5 60 0.5 61 0.5 62 0.563 0.4 64 0.4 65 0.4 66 0.3 67 0.3 68 0.3 69 0.3 70 0.2 71 0.2 72 0.2 730.2 74 0.20 75 0.1 76 0.1 77 0.04 78 0

TABLE 10 Levels of Adalimumab Expression in CHO Cells Transfected withpA205A1 pA205A1 clone# 0 nM MTX 20 nM MTX 100 nM MTX 1 4.50 11.5 18.9 22.50 9.2 17.4 3 2.40 9 15.6 4 2.10 7.4 11.4 5 2.00 6.9 10.8 6 2.00 5.810.6 7 1.90 5.6 8.6 8 1.40 5.4 7.8 9 1.40 5.3 7.1 10 1.20 4.8 5.9 111.20 4.2 5.5 12 0.90 3.6 3.2 13 0.80 2.7 0.6 14 0.70 1.8 0.5 15 0.50 1.316 0.40 0.6 17 0.30 0.6 18 0.07 0.05 19 0.00 0.03 20 0.00 0

TABLE 11 Levels of Adalimumab Expression in CHO Cells Transfected withpA205A2 pA205A2 clone# 0 nM MTX 20 nM MTX 100 nM MTX 1 9.4 32.4 26.8 26.0 21.5 17.9 3 4.80 21.2 17.7 4 4.60 19.1 15 5 4.20 18.6 13.2 6 3.7016.6 12 7 3.50 13.9 7.6 8 3.50 13.4 6.5 9 3.20 12.3 5.2 10 3.0 11.4 4.811 2.8 11.2 4.6 12 2.7 10.4 4.2 13 2.60 9.6 2.6 14 2.60 9.4 2 15 2.509.2 1.6 16 2.5 8.5 0.01 17 2.40 7.5 18 2.4 6.7 19 2.30 5.3 20 2.30 5 212.3 4.9 22 2.3 4.8 23 2.20 4.2 24 1.90 3.6 25 1.8 2.6 26 1.8 2.4 27 1.702.3 28 1.6 1.8 29 1.5 1.6 30 1.40 1.5 31 0.8 1.2 32 0.8 1.1 33 0.7 0.834 0.6 0.7 35 0.6 0.3 36 0.5 0.3 37 0.5 0.05 38 0.40 0 39 0.3 0 40 0.2 041 0.1 42 0.1 43 0.07 44 0.05 45 0.05 46 0.00 47 0

TABLE 12 Levels of Adalimumab Expression in CHO Cells Transfected withpA205A2-Spe-trunc pA205A2-Spe- trunc clone# 0 nM MTX 20 nM MTX 1 5.5 4.72 3.1 2.3 3 2.1 2.0 4 2 2.0 5 1.3 1.9 6 1.2 1.8 7 1.1 1.4 8 1.1 1.2 9 11.0 10 0.9 0.8 11 0.9 0.4 12 0.9 0.2 13 0.8 0 14 0.7 0 15 0.7 0 16 0.717 0.6 18 0.5 19 0.4 20 0.4 21 0.4 22 0.4 23 0.4 24 0.3 25 0.2 26 0.2

TABLE 13 Levels of Adalimumab Expression in CHO Cells Transfected withpA205A2-Swa-trunc pA205A2-Swa- trunc clone# 0 nM MTX 20 nM MTX 1 1.1 1.12 0.7 0.8 3 0.7 0.8 4 0.6 0.7 5 0.6 0.5 6 0.5 0.4 7 0.5 0.4 8 0.4 0.4 90.4 0.4 10 0.3 0.4 11 0.3 0.4 12 0.3 0.2 13 0.3 0.05 14 0.3 0 15 0.3 016 0.3 0 17 0.3 18 0.2 19 0.2 20 0.2 21 0.2 22 0.2 23 0.2 24 0.1 25 0.126 0.1 27 0.08 28 0

TABLE 14 Levels of Anti-EL-Selection Antibody Expression in CHO CellsTransfected with pCHOEL246GGhum pCHOEL246GGhum 0 nM 20 nM 100 nM 500 nMclone# MTX MTX MTX MTX 1 14.4 24.4 22.9 32.8 2 5.5 11.7 15.4 20.2 3 3.910.3 13.5 13.9 4 2.3 8.5 9.8 10.5 5 2.3 7.1 8.5 8.2 6 2.3 6.7 6.2 7.7 71.7 6.5 5.4 7.4 8 1.6 5.8 4.4 5.4 9 1.6 4.9 4.0 5.2 10 1.6 3.7 3.6 5.011 1.6 3.6 2.2 3.3 12 1.5 3.2 2.0 1.3 13 1.4 2.3 1.8 14 1.4 2.3 1.4 151.4 2.2 16 1.3 2.0 17 1.2 1.8 18 1.1 1.3 19 1.1 0.8 20 0.9 21 0.8 22 0.523 0.4 24 0.3 25 0.2

TABLE 15 Levels of Anti-EL-Selectin Antibody Expression in CHO CellsTransfected with pA-Gen-EL-Selectin pA-Gen-EL-Selectin 0 nM 20 nM 100 nM500 nM clone# MTX MTX MTX MTX 1 9.1 30.5 30.0 62.5 2 5.6 15.0 27.1 35.53 4.1 14.1 22.8 34.4 4 3.4 13.4 22.6 31.3 5 3.4 13.4 19.4 30.1 6 3.2 9.616.3 29.9 7 3.2 6.4 13.0 26.9 8 3.1 6.2 12.8 25.1 9 2.9 5.7 12.6 19.9 102.8 5.6 12.1 17.9 11 2.8 5.2 11.7 14.4 12 2.7 5.1 9.0 12.5 13 2.3 4.58.1 8.6 14 2.1 4.5 7.9 3.3 15 2.0 4.4 6.5 2.8 16 1.8 4.1 6.4 0.7 17 1.84.0 5.7 18 1.6 3.9 19 1.6 3.8 20 1.4 3.4 21 1.3 3.4 22 1.1 3.3 23 1.12.7 24 1.1 2.6 25 1.0 2.6 26 1.0 2.4 27 0.9 2.3 28 0.9 2.1 29 0.8 30 0.831 0.7 32 0.6 33 0.2 34 0.1

Example 8 Enhancement of Expression of Anti-IL-13 Antibody

This study shows rEVE-mediated enhancement of expression of a humanizedanti-IL-13 antibody in stably transfected mammalian cells in culturecompared to cells transfected with the expression lacking rEVE.

DNA molecules encoding the heavy and light chains of a humanizedanti-IL-13 monoclonal antibody, which has the human IgGγ1 isotype, (see,U.S. Ser. No. 11/899,819, incorporated herein by reference) wereinserted into expression vector plasmid pBJ, a DHFR-MTX amplifiableexpression plasmid, using standard methods to yield the expressionplasmid pBJ-13C5.5 (parent expression plasmid). A rEVE polynucleotidecomprising the ARM2 sequence (SEQ ID NO:2) was then inserted to plasmidpBJ-13C5.5 to yield the rEVE-containing expression plasmid pA2-13C5.5(ARM2 rEVE expression plasmid).

CHO cells were transfected with either parent expression plasmidpBJ-13C5.5 or ARM2-containing rEVE expression plasmid pA2-13C5.5following the general transfection protocol described in the previousexamples to obtain stable transfectant CHO cells. At 24 hours aftertransfection, cells from each transfection were seeded into forty-eight(48) 96-well culture plates (200 cells/well). The culture media ofindividual wells were screened by ELISA for expression of human IgG.Optical densities were slightly higher in cultures (wells) of cellstransfected with the ARM2 rEVE expression plasmid than for cellstransfected with the parent expression plasmid. An average of 74colonies (wells) per culture plate survived the selection process whencells were transfected with ARM2 rEVE plasmid pA2-13C5.5 compared to anaverage of 68 colonies per plate for cells transfected with the parentplasmid pBJ-13C5.5. Fifteen (15) stably transfected clones from eachtransfection were then subjected to DHFR-methotrexate (MTX)amplification.

Prior to amplification, the average level of expression of anti-IL-13antibody obtained from ARM2 rEVE plasmid transfectant clones wastwo-fold higher than the average level of expression from the parentplasmid transfectant clones. Antibody expression was then amplified byeither of two protocols. Using the basic protocol for DHFR-MTXamplification as described in Example 2, above, clones were firstsubjected to selection at a concentration of 20 nM MTX, followed byfurther selection at 100 nM MTX. In another amplification protocol,clones were selected directly at 100 nM MTX without an interveningselection at an intermediate MTX concentration. Table 16, below, showsthe average level of production of anti-IL-13 antibody in cultures ofthe top three producing clones from each transfection after two passagesin media containing the indicated level of amplification (concentrationof methotrexate, “MTX”).

TABLE 16 Level of Humanized Anti-IL-13 Antibody in Transfected CHO CellsExpression Vector 0 nM MTX 20 nM MTX 100 nM MTX pBJ-13C5.5 2.63 μg/ml2.00 μg/ml  3.04 μg/ml pBJ-13C5.5 2.63 μg/ml N.A.  1.72 μg/ml pA2-13C5.54.35 μg/ml 5.85 μg/ml 14.94 μg/ml pA2-13C5.5 4.35 μg/ml N.A. 14.33 μg/mlN.A. = not applicable, not part of amplification protocol

The data in Table 16 clearly show that the ARM2 rEVE enhanced the levelof expression of humanized anti-IL-13 antibody by approximately 3-foldto 5-fold compared to the level of expression obtained fromtransfectants carrying the parent expression plasmid lacking the ARM2rEVE DNA. In addition, one subclone of an ARM2 rEVE expression plasmidtransfectant expressed the humanized anti-IL-13 antibody at a level ashigh 95 μg/ml and a specific productivity of 21.74 pg/cell/day whengrown at an amplification level of 100 nM MTX.

Amplified levels of expression of anti-IL-13 antibody by clonestransfected with the ARM2 rEVE expression plasmid were readilymaintained for at least three weeks when grown under MTX selection.

Example 9 Effect of ARM1 and ARM2 rEVE Polynucleotide Molecules onExpression of Adalimumab in a Transient Expression System

This study was designed to determine whether rEVE polynucleotidemolecules will enhance levels of expression of recombinant proteins in atransient expression system.

HEK 293 cells were transfected with expression plasmid vectors pA205,pA205Genomic, pA205A1, pA205A2, pA205A2-Spec-trunc, andpA205A2-Swa-trunc. HEK293 cells cultured in Freestyle 293 ExpressionMedium (GIBCO®, Invitrogen, Carlsbad, Calif.) were transfected withplasmid DNAs complexed with polyethylenimine according to publishedconditions (Durocher et al., Nucleic Acids Res., 30: E9). No selectionfor vector integration was performed. Cells were cultured for seven daysafter transfection and aliquots of culture supernatant were tested foradalimumab concentration as described above. The results of threetransfections are shown in Table 17. The levels of expression were runin triplicate for the third transfection.

TABLE 17 Level* of Adalimumab Expression in Transfected HEK 293 CellspA205 pA205A2- pA205A2- Transfection pA205 Genomic pA205A1 pA205A2Spe-trunc Swa-trunc 1 2.1 5.4 5.4 3.3 2 5.3 20.3 20.3 9.9 3 4.3 11.911.9 10.2 2.2 1.2 4.1 15.7 15.7 2.3 1.3 3.0 14.2 14.2 2.3 1.0 *μg/ml ofadalimumab

The results in Table 17 show that ARM1 and ARM2 sequences can enhancethe level of expression of adalimumab in HEK 293 cells when presentseparately on an expression plasmid vector (pA205A1, pA205A2) or incombination (pA205Genomic). ARM1 conferred a greater level ofenhancement of adalimumab expression than ARM2 in this HEK 293 transientexpression system. In contrast, ARM2 conferred a greater level ofenhancement of expression of adalimumab than ARM1 in the CHO stableexpression system (see, Example 3, above). As seen in the CHO stableexpression system (Example 6, above), the enhancement effect of ARM2 wasalso clearly reduced in HEK 293 cells transfected with an expressionplasmid vector carrying either the SpeI- or SwaI-truncated ARM2sequence, including a lowering of expression below that seen in cellstransfected with the pA205 control (no ARM sequence) plasmid expressionvector.

All patents, applications, and publications cited in the above text areincorporated herein by reference.

Other variations and embodiments of the invention described herein willnow be apparent to those skilled in the art, and all such variants andalternative embodiments of the invention are intended to be encompassedwithin the foregoing description and the claims that follow.

1. A yeast host cell comprising a recombinant vector, wherein therecombinant vector comprises a recombinant expression vector element(rEVE) polynucleotide molecule comprising a nucleotide sequence selectedfrom the group consisting of: the nucleotide base sequence of SEQ IDNO:1, the nucleotide base sequence of SEQ ID NO:2, a sequencecomplementary to any foregoing sequence, an expression-enhancing portionof any foregoing sequence, and combinations thereof; and wherein theyeast host cell is selected from the group consisting of: Hansenulapolymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomycespombe, and Yarrowia lipolytica.
 2. A nematode host cell comprising arecombinant vector, wherein the recombinant vector comprises arecombinant expression vector element (rEVE) polynucleotide moleculecomprising a nucleotide sequence selected from the group consisting of:the nucleotide base sequence of SEQ ID NO:1, the nucleotide basesequence of SEQ ID NO:2, a sequence complementary to any foregoingsequence, an expression-enhancing portion of any foregoing sequence, andcombinations thereof; and wherein the nematode host cell isCaenorhabditis elegans.
 3. A prokaryotic host cell comprising arecombinant vector, wherein the recombinant vector comprises arecombinant expression vector element (rEVE) polynucleotide moleculecomprising a nucleotide sequence selected from the group consisting of:the nucleotide base sequence of SEQ ID NO:1, the nucleotide basesequence of SEQ ID NO:2, a sequence complementary to any foregoingsequence, an expression-enhancing portion of any foregoing sequence, andcombinations thereof; and wherein the prokaryotic host cell is selectedfrom the group consisting of: Escherichia coli, a serovar of Salmonellaenterica, a Shigella species, Wollinella succinogenes, Proteus vulgaris,Proteus mirabilis, Edwardsiella tarda, Citrobacter freundii, aPasteurella species, a Haemophilus species, a Pseudomonas species, aBacillus species, a Staphyloccocus species, and a Streptococcus species.4. A method of producing a recombinant protein of interest comprising:inserting into a host cell a recombinant expression vector wherein therecombinant expression vector comprises: one or more genes encoding therecombinant protein of interest; and a nucleotide base sequence selectedfrom the group consisting of: the nucleotide base sequence of SEQ IDNO:1, the nucleotide base sequence of SEQ ID NO:2, a sequencecomplementary to any foregoing sequence, an expression-enhancing portionof any foregoing sequence, and combinations thereof; and culturing thehost cell under conditions promoting expression of the recombinantprotein of interest.
 5. The method according to claim 4, wherein saidrecombinant protein of interest is selected from the group consisting ofa soluble protein, a membrane protein, a structural protein, a ribosomalprotein, an enzyme, a zymogen, an antibody molecule, a cell surfacereceptor protein, a transcription regulatory protein, a translationregulatory protein, a chromatin protein, a hormone, a cell cycleregulatory protein, a G protein, a neuroactive peptide, animmunoregulatory protein, a blood component protein, an ion gateprotein, a heat shock protein, a dihydrofolate reductase (DHFR), anantibiotic resistance protein, a functional fragment of any of thepreceding proteins, an epitope-containing fragment of any of thepreceding proteins, and combinations thereof.
 6. The method according toclaim 5, wherein the antibody molecule is selected from the groupconsisting of an antibody to an immunoregulatory protein, an antibody toa selectin, an antibody to a cell surface receptor, and a dual variabledomain immunoglobulin (DVD-Ig) molecule.
 7. The method according toclaim 6, wherein the antibody to an immunoregulatory protein is ananti-IL-13 antibody or an anti-TNF-α antibody.
 8. The method accordingto claim 7, wherein the anti-TNF-α antibody is adalimumab.
 9. The methodaccording to claim 6, wherein the antibody molecule is a dual variabledomain immunoglobulin (DVD-Ig) molecule.
 10. The method according to anyone of claims 4-9, wherein the recombinant expression vector comprisesat least one copy of a gene encoding a dihydrofolate reductase.
 11. Themethod according to any one of claims 4-9, wherein the host cell isselected from the group consisting of: a Chinese Hamster Ovary (CHO)cell, a COS cell, a Vero cell, an SP2/0 cell, an NS/0 myeloma cell, ahuman embryonic kidney (HEK 293) cell, a baby hamster kidney (BHK) cell,a HeLa cell, a human B cell, a CV-1/EBNA cell, an L cell, a 3T3 cell, anHEPG2 cell, a PerC6 cell, and an MDCK cell.
 12. The method according toclaim 11, wherein the recombinant expression vector comprises at leastone copy of a gene encoding a dihydrofolate reductase.